CRISPR/Cas systems for the detection of nucleic acid and non-nucleic acid targets.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are becoming powerful tools for disease biomarkers detection. Due to the specific recognition, cis-cleavage and nonspecific trans-cleavage capabilities, CRISPR/Cas systems have implemented the detection of nucleic acid targets (DNA and RNA) as well as non-nucleic acid targets (e.g., proteins, exosomes, cells, and small molecules). In this review, we first summarize the principles and characteristics of various CRISPR/Cas systems, including CRISPR/Cas9, Cas12, Cas13 and Cas14 systems. Then, various types of applications of CRISPR/Cas systems used in detecting nucleic and non-nucleic acid targets are introduced emphatically. Finally, the prospects and challenges of their applications in biosensing are discussed.

Ultrasensitive DNA-Biomacromolecule Sensor for the Detection Application of Clinical Cancer Samples.

Diagnostic testing of biological macromolecules is of great significance for early warning of disease and cancer. Nevertheless, restricted by limited surface area and large steric hindrance, sensitive detection of macromolecules with interface-based sensing method remains challenging. Here, a "biphasic replacement" electrochemical aptamer-based (BRE-AB) sensing strategy which placed capture reaction of the biomacromolecule in a homogeneous solution phase and replaced with a small diameter of single-stranded DNA to attach to the interface is introduced. Using the BRE-AB sensor, the ultrasensitive detection of luteinizing hormone (LH) with the detection limit of 10 × 10-12 m is demonstrated. Molecular Dynamics simulations are utilized to explore the binding mechanism of aptamer and target LH. Moreover, it is confirmed that the BRE-AB sensor has excellent sensing performance in whole blood and undiluted plasma. Using the BRE-AB sensor, the LH concentrations in 40 clinical samples are successfully quantified and it is found that LH is higher expressed in breast cancer patients. Furthermore, the sensor enables simple, low-cost, and easy to regenerate and reuse, indicating potentially applicable for point-of-care biological macromolecules diagnostics.

Nonplanar Helicene Benzo[4]Helicenium for the Precise Treatment of Renal cell Carcinoma.

Immune and targeted therapy are becoming the first-line treatment for renal cell carcinoma (RCC). However, therapeutic outcomes are limited due to the low efficiency and side effect. Here, it is found that helicenes are able to exhibit an anticancer capability through changing the molecular structure from planar to nonplanar. Furthermore, the cytotoxicity in vitro and cancer inhibition ability of nonplanar helicenes increase with its aromatic rings' number. It is further demonstrated that benzo[4]helicenium shows the specific killing efficiency against the RCC cancer as compared to normal kidney cells. This is majorly originated from a more selective damage of benzo[4]helicenium for mitochondria and DNA in RCC cancer cells, not the normal kidney. The selective killing ability of benzo[4]helicenium makes it have potential to be used as a targeted drug for the precise treatment of RCC.

P2X7 receptor signaling promotes inflammation in renal parenchymal cells suffering from ischemia-reperfusion injury.

Extracellular adenosine triphosphate (ATP) and its receptor, P2X7 receptor (P2X7R), are playing an important role in the pathological process of renal ischemia-reperfusion injury, but their underlying mechanism remains unclear. Also, the effects of tubular epithelium-expressed P2X7 receptor on ischemia acute kidney injury is still unknown. The aim of this study is to clarify if this mechanism involves the activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in the renal tubular epithelial cells. In our research, we used male C57BL/6 wild type and P2X7R (-/-) mice, cultured human proximal tubular epithelial cells, and kidneys from acute kidney injury patients. Mice underwent for unilateral nephrectomy combined with the lateral renal pedicle clamping. Cultured cells were subjected to hypoxia/reoxygenation or ATP. Apyrase and A438079 were used to block the extracellular ATP/P2X7 receptor pathway. We also constructed radiation-induced bone marrow (BM) chimeras by using P2X7R (-/-) mice and P2X7R (+/+) wild-type mice. P2X7 receptor deficiency protected from renal ischemia-reperfusion injury and attenuated the formation of NLRP3 inflammasome. By using BM chimeras, we found a partial reduction of serum creatinine and less histological impairment in group wild-type BM to P2X7R (-/-) recipient, compared with group wild-type BM to wild-type recipient. In renal tubular epithelial cells, hypoxia/reoxygenation induced ATP release and extracellular ATP depletion reduced the expression of active IL-1ß. ATP activated the NLRP3 inflammasome in renal tubular epithelial cells, which were blunted by transient silence of P2X7 receptor, as well as by P2X7 receptor blocking with A438079. In human samples, we found that patients with Stage 3 AKI had higher levels of P2X7 receptor expression than patients with Stage 1 or Stage 2. Extracellular ATP/P2X7 receptor axis blocking may protect renal tubular epithelial cells from ischemia-reperfusion injury through the regulation of NLRP3 inflammasome.

Differential toxicity mechanism of gold nanoparticles in HK-2 renal proximal tubular cells and 786-0 carcinoma cells.

Aim: To research the influence and mechanism of gold nanoparticles (AuNPs) with different size for HK-2 cells (kidney normal cells) and 786-0 cells (kidney cancer cells). Materials & methods: HK-2 cells and 786-0 cells were treated with 5 and 200 nm AuNPs at 1 and 10 µg/ml. The cell viability, intracellular reactive oxygen species levels, cell apoptosis, cell autophagy, and related cell signaling pathways were analyzed. Results: In HK-2 cells, AuNPs reduced the activity of Akt and mTOR and upregulated the expression of LC3 II. In 786-0 cells, the activity of p38 was upregulated, which leaded to the increase of caspase 3 and initiated apoptosis. Conclusion: AuNPs of 5 and 200 nm at 10 µg/ml exerted antitumor effect by prompting apoptosis and inhibiting proliferation, while autophagy was activated to protect HK-2 cells from AuNPs-induced cytotoxicity.

Programing Assembling/Releasing Multifunctional miRNA Nanomedicine to Treat Prostate Cancer.

MicroRNAs (miRNAs) therapy has shown to have great promise for the treatment of androgen-independent prostate cancer (AIPC) due to the low efficiency of hormonal therapy. However, instability of RNA and inefficiency of RNA therapy limit the use of miRNAs in the treatment of AIPC. Here, we report a pH/ATP-activated nanocomplexes for increasing cytosolic delivery of miR146a which can effectively inhibit the expression of epidermal growth factor receptor (EGFR) in AIPC. The nanocomplexes show identical suppressing effect in invasion, colony formation, migration ability, and growth of DU145 cells compared with Lipofectamine 2000 (lipo). But for in vivo experiments, the nanocomplexes vigorously suppress the growth of tumor volumes comparing to lipo group after five weeks' treatment. These results demonstrate the potential of the pH/ATP-activated nanocarriers for AIPC gene therapy.

Reduction of mitochondria and up regulation of pyruvate dehydrogenase kinase 4 of skeletal muscle in patients with chronic kidney disease.

AIM: Muscle weakness is commonly among chronic kidney disease (CKD) patients. Muscle mitochondrial dysfunction and decreased pyruvate dehydrogenase (PDH) activity occur in CKD animals but have not been confirmed in humans, and changes in pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP) expression have not been evaluated in CKD muscle. We presume that the reduction of muscle mitochondria and post-translational modification of PDH may cause muscle weakness in CKD patients. Herein, we explored changes in mitochondrial morphology, PDH expression and activity, and PDK/PDP expression in CKD patient muscle. METHODS: Twenty patients with stage 4-5 CKD (CKD group) and 24 volunteers (control group) were included. Clinical characteristics, biochemical information and handgrip strength (HGS) were determined. Skeletal muscle samples were collected from eight stage 5 CKD patients from CKD group. Other eight non-CKD surgical subjects' muscle samples were collected as control. PDH activity was determined using a PDH enzyme activity assay kit, and real-time PCR and western blotting analyses were performed to measure gene expression and protein levels, respectively. Transmission electron microscopy was used to study mitochondria morphology. RESULTS: CKD patients had lower HGS than non-CKD subjects, and HGS was correlated with gender, age, haemoglobin and albumin. Mitochondria were decreased in end-stage renal disease (ESRD) patients muscle. Mfn-1 expression and phospho-Drp1(S637)/Drp1 ratio were inhibited in the ESRD group, implicating dysfunctional mitochondrial dynamics. Muscle PDH activity and phospho-PDH(S293) were decreased in ESRD patient muscle, while PDK4 protein level was up regulated. CONCLUSION: Decreased mitochondria and PDH deficiency caused by up regulation of PDK 4 contribute to muscle dysfunction, and could be responsible for muscle weakness in CKD patients.

Yes-associated protein regulates podocyte cell cycle re-entry and dedifferentiation in adriamycin-induced nephropathy.

Podocytes are terminally differentiated cells with little proliferative capacity. The high expression levels of cell cycle inhibitory proteins, including p21, p27, and p57, play an important role in maintaining the low level of proliferation of mature podocytes. In the present study, we aimed to explore the role of yes-associated protein (YAP) signalling in adriamycin-induced podocyte re-entry into the cell cycle and dedifferentiation. Proliferating cell nuclear antigen (PCNA)-, cyclin-dependent kinase 4 (CDK4)-, and Cyclin D1-positive podocytes were found in mice with adriamycin-induced nephropathy. In vitro, adriamycin administration increased the percentage of cells in S phase and the upregulation of mesenchymal-related marker proteins. CDK4 and cyclin D1 were significantly up-regulated after incubation with adriamycin. Overexpression of YAP in podocytes promoted their entry into the cell cycle; up-regulated cyclin D1, desmin, and snail2 expression and down-regulated Wilms' tumour 1 (WT1) and nephrin production. Recombinant murine FGF-basic induced podocytes to re-enter the cell cycle, inhibited WT1 and nephrin, and increased desmin and snail2 expression. Pretreating podocytes with verteporfin, an inhibitor of YAP/ TEA domain transcription factor (TEAD), decreased the adriamycin-induced overexpression of cyclin D1 and reduced the ratio of S-phase podocytes. This result was further verified by knocking down YAP expression using RNA interference. In conclusion, adriamycin induced podocytes to re-enter the cell cycle via upregulation of CDK4 and cyclin D1 expression, which was at least partly mediated by YAP signalling. Re-entry into the cell cycle induced the over-expression of mesenchymal markers in podocytes.

Serum miR-192 Is Related to Tubulointerstitial Lesion and Short-Term Disease Progression in IgA Nephropathy.

BACKGROUND/AIMS: Clinical manifestation and renal histology serve as the current "gold standard" for evaluation of renal lesions in IgA nephropathy. MiR-192 is regarded as a potential noninvasive biomarker for kidney disease. We sought to elucidate a relationship between intrarenal, serum, and urinary exosomal miR-192 with renal lesions and disease progression in IgA nephropathy. METHODS: Serum and urinary exosomal miR-192 were detected and correlated with clinical and pathological parameters from consecutive IgA nephropathy patients (n = 50) and healthy control patients (n = 25). Patients then received a follow-up of 24 months after biopsy. Disease progression was defined as a 40% reduction in estimated glomerular filtration rate. Expression of miR-192 was quantified by Taqman RT-PCR. Intrarenal -miR-192 was detected in 8 IgA nephropathy patients and 4 matched patients receiving kidney nephrectomy as controls via in situ hybridization. TGF-ß1 and E-cadherin expression in renal tissue was evaluated using immunohistochemistry. RESULTS: Intrarenal miR-192 was correlated with serum miR-192 (r = 0.690, p = 0.013). Both intrarenal and serum miR-192 decreased in IgA nephropathy patients and were correlated with estimated glomerular filtration ratio. Patients with lower intrarenal and serum miR-192 levels had a higher interstitial fibrosis and tubular atrophy score, more severe lesions in tubular atrophy, and interstitial inflammation. Renal E-cadherin expression was correlated (r = 0.484, p = 0.004) and TGF-ß1 was inversely correlated (r = -0.527, p < 0.001) with serum miR-192 in IgA. Patients with higher serum miR-192 had a lower disease progression rate over the course of 2 years (0 vs. 16%, p = 0.028). No correlation was found in urinary exosomal miR-192 with the clinical and pathological parameters mentioned earlier. CONCLUSIONS: Lower serum miR-192 in IgA nephropathy patients indicates lower intrarenal miR-192 expression, more severe tubular atrophy, interstitial inflammation, and fibrotic tendency (with higher TGF-ß and E-cadherin in renal miR-192). IgA nephropathy patients with higher serum miR-192 are less likely to have renal function decline over the course of 2 years.

Is Pooled CRISPR-Screening the Dawn of a New Era for Functional Genomics.

Functional genomics aims to develop an in-depth understanding of how specific gene dysfunctions are related to diseases. A common method for investigating the genome and its complex functions is via perturbation of the interactions between the DNA, RNA and their protein respective protein derivatives. Commonly, arrayed and pooled genetic screens are utilized to achieve this and in recent years have been fundamental in achieving the current level of understanding for gene dysfunctions. However, they are limited in specific aspects which scientists have attempted to address. Clustered regularly palindromic repeats (CRISPR)-based methods for genetic screens have in recent years become more prevalent but crucially shared similar properties to previous methods and failing to provide a distinct advantage over previous methods. CROP-seq, Perturb-seq, and CRISPR-seq have combined CRISPR and single-cell RNA-sequencing (scRNA-seq) and is the newest addition to the geneticist's arsenal, providing scientists with methods to edit DNA with improved speed, accuracy, and efficiency which could usher us into a new era of study methods for functional genomics. We briefly overview the CRISPR-Cas9 systems, the evolution of genetic screening in recent years, and evaluate and discuss the significance of CROP-seq, Perturb-seq, and CRISPR-seq.

Pharmacological inhibition of heparin-binding EGF-like growth factor promotes peritoneal angiogenesis in a peritoneal dialysis rat model.

BACKGROUND: Molecular mechanisms of peritoneal dialysis (PD) ultrafiltration failure, peritoneal neo-angiogenesis, and fibrosis remain to be determined. We aimed to determine the role of heparin-binding EGF-like growth factor (HB-EGF) inhibition on angiogenesis of peritoneal membrane in a PD rat model. METHODS: 32 male Wistar rats were assigned into (1) control group; (2) uremic non-PD group: subtotal nephrectomy-induced uremic rats without PD; (3) uremic rats subjected to PD: uremic rats that were dialyzed with Dianeal® for 4 weeks; (4) CRM 197 group: dialyzed uremic rats were supplemented with CRM197, a specific HB-EGF inhibitor. Peritoneal transport function was examined by peritoneal equilibration test. Expression of HB-EGF and EGFR in peritoneal samples were examined by real-time PCR, immunohistochemical staining, and western blot. RESULTS: Progressive angiogenesis and fibrosis were observed in uremic PD rats, and there were associated with decreased net ultrafiltration (nUF), increased permeability of peritoneal membrane, and reduced expression of HB-EGF and EGFR protein and mRNA in uremic PD rats compared to uremic non-PD or control groups (both p < 0.05). CRM197 significantly induced peritoneal membrane permeability, decreased nUF, increased higher vessel density, and reduced pericyte count compared to that of uremic PD rats. The levels of HB-EGF and EGFR expression negatively correlated with vessel density in peritoneal membrane (both p < 0.001). CONCLUSION: PD therapy was associated with peritoneal angiogenesis, functional deterioration, and downregulation of HB-EGF/EGFR. Pharmacological inhibition of HB-EGF promoted PD-induced peritoneal angiogenesis and fibrosis and ultrafiltration decline, suggesting that HB-EGF downregulation contributes to peritoneal functional deterioration in the uremic PD rat model.

Fe3O4 nanoparticles and cryoablation enhance ice crystal formation to improve the efficiency of killing breast cancer cells.

The key problem of cryoablation is that only freezing is often unable to kill the capillaries at tumor edges, leading to a high rate of recurrence. Here, we found that Fe3O4 nanoparticles were highly useful to improve the freezing capability of cryosurgery due to their ability to alter intracellular ice formation (IIF) and growth in tumor cells. The killing efficiency of cryoablation for MCF-7 breast cancer cells can be expected to be enhanced as the Fe3O4 nanoparticles concentration increased, it was mainly because that more IIF was induced by the participation of Fe3O4 nanoparticles during freezing, recrystallization and thawing. Furthermore, our results also showed that recrystallization contributed to the formation of extracellular embryonic crystals, which was capable of enhancing the efficiency of killing MCF-7 cells. This research is to develop an understanding of the mechanism of the cryoablation enhancing the killing efficiency in the presence of the Fe3O4 nanoparticles, and to promote their further application in tumor therapy.

Implications of dynamic changes in miR-192 expression in ischemic acute kidney injury.

PURPOSE: Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) with poor outcomes. While many important functions of microRNAs (miRNAs) have been identified in various diseases, few studies reported miRNAs in acute kidney IRI, especially the dynamic changes in their expression and their implications during disease progression. METHODS: The expression of miR-192, a specific kidney-enriched miRNA, was assessed in both the plasma and kidney of IRI rats at different time points after kidney injury and compared to renal function and kidney histological changes. The results were validated in the plasma of the selected patients with AKI after cardiac surgery compared with those matched patients without AKI. The performance characteristics of miR-192 were summarized using area under the receiver operator characteristic (ROC) curves (AUC-ROC). RESULTS: MiRNA profiling in plasma led to the identification of 42 differentially expressed miRNAs in the IRI group compared to the sham group. MiR-192 was kidney-enriched and chosen for further validation. Real-time PCR showed that miR-192 levels increased by fourfold in the plasma and decreased by about 40% in the kidney of IRI rats. Plasma miR-192 expression started increasing at 3 h and peaked at 12 h, while kidney miR-192 expression started decreasing at 6 h and remained at a low level for 7 days after reperfusion. Plasma miR-192 level in patients with AKI increased at the time of ICU admission, was stable for 2 h and decreased after 24 h. AUC-ROC was 0.673 (95% CI: 0.540-0.806, p = 0.014). CONCLUSIONS: Plasma miR-192 expression was induced in a time-dependent manner after IRI in rats and patients with AKI after cardiac surgery, comparably to the kidney injury development and recovery process, and may be useful for the detection of AKI.

Size-dependent cytotoxicity of Fe3O4 nanoparticles induced by biphasic regulation of oxidative stress in different human hepatoma cells.

The application of Fe3O4 nanoparticles (NPs) has made great progress in the diagnosis of disease and in the drug delivery system for cancer therapy, but the relative mechanisms of potential toxicity induced by Fe3O4 have not kept pace with its development in the application, which has hampered its further clinical application. In this article, we used two kinds of human hepatoma cell lines, SK-Hep-1 and Hep3B, to investigate the cytotoxic effects and the involved mechanisms of small Fe3O4 NPs with different diameters (6 nm, 9 nm, and 14 nm). Results showed that the size of NPs effectively influences the cytotoxicity of hepatoma cells: 6 nm Fe3O4 NPs exhibited negligible cytotoxicity and 9 nm Fe3O4 NPs affected cytotoxicity via cellular mitochondrial dysfunction and by inducing necrosis mediated through the mitochondria-dependent intracellular reactive oxygen species generation. Meanwhile, 14 nm Fe3O4 NPs induced cytotoxicity by impairing the integrity of plasma membrane and promoting massive lactate dehydrogenase leakage. These results explain the detailed mechanism of different diameters of small Fe3O4 NPs-induced cytotoxicity. We anticipate that this study will provide different insights into the cytotoxicity mechanism of Fe3O4 NPs, so as to make them safer to use in clinical application.

Autophagy protects renal tubular cells against ischemia / reperfusion injury in a time-dependent manner.

BACKGROUND/AIMS: Autophagy is a dynamic catabolic process that maintains cellular homeostasis. Whether it plays a role in promoting cell survival or cell death in the process of renal ischemia/reperfusion (I/R) remains controversial, partly because renal autophagy is usually examined at a certain time point. Therefore, monitoring of the whole time course of autophagy and apoptosis may help better understand the role of autophagy in renal I/R. METHODS: Autophagy and apoptosis were detected after mice were subjected to bilateral renal ischemia followed by 0-h to 7-day reperfusion, exposure of TCMK-1 cells to 24-h hypoxia, and 2 to 24-h reoxygenation. The effect of autophagy on apoptosis was assessed in the presence of autophagy inhibitor 3-methyladenine (3-MA) and autophagy activator rapamycin. RESULTS: Earlier than apoptosis, autophagy increased from 2-h reperfusion, reached the maximum at day 2, and then began declining from day 3 when renal damage had nearly recovered to normal. Exposure to 24-h hypoxia induced autophagy markedly, but it decreased drastically after 4 and 8-h reoxygenation, which was accompanied with increased cell apoptosis. Inhibition of autophagy with 3-MA increased the apoptosis of renal tubular cells during I/R in vivo and hypoxia/reoxygenation (H/R) in vitro. In contrast, activation of autophagy by rapamycin significantly alleviated renal tissue damage and tubular cell apoptosis in the two models. CONCLUSION: Autophagy was induced in a time-dependent manner and occurred earlier than the onset of cell apoptosis as an early response that played a renoprotective role during renal I/R and cell H/R. Up-regulation of autophagy may prove to be a potential strategy for the treatment of acute kidney injury.

Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells.

Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells.

Survey of acute kidney injury and related risk factors of mortality in hospitalized patients in a third-level urban hospital of Shanghai.

OBJECTIVE: The main aim of this study is to investigate the incidence and prognosis of acute kidney injury (AKI) and to clarify the risk factors associated with the prognosis of AKI in hospitalized patients. METHOD: All patients hospitalized from January 1st to December 31st 2012 in Ren Ji Hospital, School of Medicine Shanghai Jiao Tong University were screened by the Lab Administration Network. All the patients with an intact medical history of AKI according to the Acute Kidney Injury Network (AKIN) were enrolled in the study cohort. AKI's incidence and etiology, as well as the patient's characteristics and prognosis, were retrospectively analyzed. Logistic regression analysis was used to investigate the risk factors on the patient prognosis and renal outcome. RESULTS: 934 AKI patients were enrolled. The incidence of AKI in hospitalized patients was 2.41%. The ratio of males to females of patients was 1.88:1 and the mean age was 60.82 ± 16.94. The incidence of AKI increased with increase in age. Among hospitalized patients, 63.4% were from the surgical department, 35.4% from the internal medicine department, and 1.2% from the obstetric and gynecologic department. Regarding the cause of AKI, pre-renal AKI, acute tubular necrosis (ATN), acute glomerulonephritis and vasculitis (AGV), acute interstitial nephritis (AIN), and post-renal AKI contributed with 51.7, 37.7, 3.8, 3.5, and 3.3%, respectively. The survival rate on the day 28 after AKI was 71.8%. In addition, 65.7% patients got complete renal recovery, while 16.9% got partial renal recovery and 17.4% got renal loss. The mortality of AKI in hospitalized patients at Stage I, Stage II and Stage III was 24.8, 31.2 and 43.7%, respectively. Multivariate Logistic regression analysis showed that use of nephrotoxic drugs, [Odds Ratio (OR) = 2.313], hypotension in the previous week (OR = 4.482), oliguria (OR = 5.267), the number of extra-renal organ failures (OR = 1.376), and need for renal replacement therapy (RRT) (OR = 4.221) were independent risk factors for mortality. The number of extra-renal organ failures (OR = 1.529) and RRT (OR = 2.117) were independent risk factors for renal loss. CONCLUSION: AKI is one of the most common complications in hospitalized patients. The mortality is high and renal prognosis is poor after AKI. The prognosis is closely associated with the severity of AKI. Nephrotoxic drugs, hypotension within the last week, oliguria, the number of extra-renal organ failures, and RRT are independent risk factors for mortality, while the number of extra-renal organ failures and RRT are independent risk factors for renal loss.

Renal tissue thawed for 30 minutes is still suitable for gene expression analysis.

Some biosamples obtained from biobanks may go through thawing before processing. We aim to evaluate the effects of thawing at room temperature for different time periods on gene expression analysis. A time course study with four time points was conducted to investigate the expression profiling on 10 thawed normal mice renal tissue samples through Affymetrix GeneChip mouse gene 2.0 st array. Microarray results were validated by quantitative real time polymerase chain reactions (qPCR) on 6 candidate reference genes and 11 target genes. Additionally, we used geNorm plus and NormFinder to identify the most stably expressed reference genes over time. The results showed RNA degraded more after longer incubation at room temperature. However, microarray results showed only 240 genes (0.91%) altered significantly in response to thawing at room temperature. The signal of majority altered probe sets decreased with thawing time, and the crossing point (Cp) values of all candidate reference genes correlated positively with the thawing time (p<0.05). The combination of B2M, ACTB and PPIA was identified as the best choice for qPCR normalization. We found most target genes were stable by using this normalization method. However, serious gene quantification errors were resulted from improper reference genes. In conclusion, thirty minutes of thawing at room temperature has a limited impact on microarray and qPCR analysis, gene expression variations due to RNA degradation in early period after thawing can be largely reduced by proper normalization.

Impact of thawing on reference gene expression stability in renal cell carcinoma samples.

More and more samples are obtained from biobanks for biomedical research; however, some of these samples may undergo thawing before processing. We aim to evaluate the reference gene expression stability in thawed renal cell carcinoma samples. Sixteen matched malignant and nonmalignant renal tissue samples were obtained and each sample was divided into 4 aliquots before being snap frozen and stored at -80°C. By quantitative real-time polymerase chain reaction, a time-course study was conducted on the thawed tissue to evaluate the expression stability of a panel of the 10 most frequently used reference genes in renal cell carcinom samples: ACTB, ALAS1, B2M, GAPDH, HMBS, HPRT, PPIA, RPLP0,TBP, and TUBB. As shown by geNorm M values, PPIA was the most stable gene at the 0-, 15-, and 30-minute time points (M=0.82, 0.85, and 0.76, respectively), whereas GAPDH was ranked last at the 5-, 15-, and 30-minute time points (M=1.38, 1.44, and 1.39, respectively). A positive correlation was found by linear regression between the thawing time and 2 to the power of crossing point values of all candidate reference genes (P<0.05). The mean coefficient of variance of all reference genes increased significantly at time points 5, 15, and 30 minutes compared with 0 minutes (P<0.01). In conclusion, using the geNorm algorithm, PPIA was identified as the most stably expressed gene between malignant and nonmalignant renal tissue samples that were thawed for similar time periods. All the reference genes showed high variations along with the thawing time; it should be recommended to use a combination of several candidate reference genes when comparing samples thawed for different time periods.

Consent for use of clinical leftover biosample: a survey among Chinese patients and the general public.

BACKGROUND: Storage of leftover biosamples generates rich biobanks for future studies, saving time and money and limiting physical impact to sample donors. OBJECTIVE: To investigate the attitudes of Chinese patients and the general public on providing consent for storage and use of leftover biosamples. DESIGN, SETTING AND PARTICIPANTS: Cross-sectional surveys were conducted among randomly selected patients admitted to a Shanghai city hospital (n = 648) and members of the general public (n = 492) from May 2010 to July 2010. MAIN OUTCOME MEASURES: Face-to-face interviews collected respondents-report of their willingness to donate residual biosample, trust in medical institutions, motivation for donation, concerns of donated sample use, expectations for research results return, and so on. RESULTS: The response rate was 83.0%. Of the respondents, 89.1% stated that they completely understood or understood most of questions. Willingness to donate residual sample was stated by 64.7%, of which 16.7% desired the option to withdraw their donations anytime afterwards. Only 42.3% of respondents stated they "trust" or "strongly trust" medical institutions, the attitude of trusting or strongly trusting medical institutions were significantly associated with willingness to donate in the general public group.(p<0.05) The overall assent rate for future research without specific consents was also low (12.1%). Hepatitis B virus carriers were significantly less willing than non-carriers to donate biosamples (32.1% vs. 64.7%, p<0.001). CONCLUSIONS: Low levels of public trust in medical institutions become serious obstacle for biosample donation and biobanking in China. Efforts to increase public understanding of human medical research and biosample usage and trust in the ethical purposes of biobanking are urgently needed. These efforts will be greatly advanced by the impending legislation on biobanking procedures and intent, and our results may help guide the structure of such law.