A faster and less costly alternative for RNA extraction of SARS-CoV-2 using proteinase k treatment followed by thermal shock.

One of the biggest challenges during the pandemic has been obtaining and maintaining critical material to conduct the increasing demand for molecular tests. Sometimes, the lack of suppliers and the global shortage of these reagents, a consequence of the high demand, make it difficult to detect and diagnose patients with suspected SARS-CoV-2 infection, negatively impacting the control of virus spread. Many alternatives have enabled the continuous processing of samples and have presented a decrease in time and cost. These measures thus allow broad testing of the population and should be ideal for controlling the disease. In this sense, we compared the SARS-CoV-2 molecular detection effectiveness by Real time RT-PCR using two different protocols for RNA extraction. The experiments were conducted in the National Institute of Health (INS) from Peru. We compared Ct values average (experimental triplicate) results from two different targets, a viral and internal control. All samples were extracted in parallel using a commercial kit and our alternative protocol-samples submitted to proteinase K treatment (3 µg/µL, 56°C for 10 minutes) followed by thermal shock (98°C for 5 minutes followed by 4°C for 2 minutes); the agreement between results was 100% in the samples tested. In addition, we compared the COVID-19 positivity between six epidemiological weeks: the initial two in that the Real time RT-PCR reactions were conducted using RNA extracted by commercial kit, followed by two other using RNA obtained by our kit-free method, and the last two using kit once again; they did not differ significantly. We concluded that our in-house method is an easy, fast, and cost-effective alternative method for extracting RNA and conducing molecular diagnosis of COVID-19.

Assessment of Urothelial Cytotoxicity at Morphological and Molecular Levels: Urinary Bladder as Target Organ.

The urinary bladder is a target organ of several toxic agents. Exposure to those agents induces mild-to-severe changes, which can be evaluated by different methods. Among them, the scanning-electron microscopy (SEM) is the "gold standard" for characterizing urothelial damage since it provides high-definition images, making it possible to detect early lesions on the surface of the urinary bladder. In addition, molecular technologies allow detecting changes in genetic material and investigating the interaction between genes and environmental stress in disease causation. The urinary bladder epithelium is where the most common type of bladder cancer occurs in humans, that is, the transitional-cell carcinoma (TCC). In animal models, the TCC can be similar to the disease in humans. Techniques to evaluate urothelium in experimental models aid in the comprehension of risk factors for urothelial carcinogenesis.

A low-cost smart system for electrophoresis-based nucleic acids detection at the visible spectrum.

Nucleic acid detection by electrophoresis is still a quick and accessible technique for many diagnosis methods, primarily at research laboratories or at the point of care units. Standard protocols detect DNA/RNA molecules through specific bound chemical dyes using a UV-transilluminator or UV-photo documentation system. However, the acquisition costs and availability of these devices, mainly the ones with photography and internet connection capabilities, can be prohibitive, especially in developing countries public health units. Also, ultraviolet radiation is a common additional risk factor to professionals that use electrophoresis-based nucleic acid detection. With that in mind, this work describes the development of a low-cost DNA/RNA detection smart system capable of obtaining qualitative and semi-quantitative data from gel analysis. The proposed device explores the visible light absorption range of commonly used DNA/RNA dyes using readily available parts, and simple manufacturing processes, such as light-emitting diodes (LEDs) and 3D impression. By applying IoT techniques, our system covers a wide range of color spectrum in order to detect bands from various commercially used dyes, using Bluetooth communication and a smartphone for hardware control, image capturing, and sharing. The project also enables process scalability and has low manufacturing and maintenance costs. The use of LEDs at the visible spectrum can achieve very reproducible images, providing a high potential for rapid and point-of-care diagnostics as well as applications in several fields such as healthcare, agriculture, and aquaculture.

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue.

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 °C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

Assessment of Protein Profiles of RNAlater Stored and Fresh PBMC Cells Using Different Protein Extraction Buffers.

For proteome analyses, the tissue samples are mostly preserved either snap frozen or formalin-fixed, paraffin-embedded form. Use of RNAlater-a non-toxic solution primarily used to stabilize the RNA content of samples-in tissue preservation for proteome analysis recently described equally reliable with snap-frozen preservation in human tissues. Even though RNALater storage has great potential in the preservation of Peripheral Blood Mononuclear Cells (PBMC), its impact on the results of proteome analysis is poorly described at qualitative and quantitative measures. The present study investigated protein profiles of RNAlater preserved and fresh PBMCs using three extraction buffers viz. Triton X-100, RIPA and SDS. Proteins are separated in SDS-PAGE and quantified using densitometry. On an average 19.3 bands from fresh and 15.6 bands from RNAlater storage cells were obtained with a molecular weight ranging from 25 to > 250 kDa. RNAlater storage generated a fewer number and lesser quantity of low molecular weight proteins while yielded a similar or high quantity of high molecular weight protein fractions. The principal component analysis showed that Triton X-100 is inferior as compared to SDS and RIPA with respect to their protein bands and quantity yielded. While RNAlater is effective in preserving PBMC for proteome analysis, our findings warrant caution in its use in proteomics experiments especially if the target is low molecular weight proteins.

Multiplexed Digital Detection of B-Cell Acute Lymphoblastic Leukemia Fusion Transcripts Using the NanoString nCounter System.

Chromosomal rearrangements resulting in fusion transcripts have been reported in precursor B-cell acute lymphoblastic leukemia (B-ALL). The identification of fusion events is crucial in the diagnosis of B-ALL. In this study, we used NanoString nCounter technology to design, validate, and evaluate a multiplex panel for the detection of B-ALL fusion transcripts. Fifty-one B-ALL fusion transcripts reported in children in the literature were included in the design of the NanoString panel. Twenty-six fusion transcripts were validated using 64 positive-control samples and 74 negative-control samples with 100% sensitivity and 99% specificity in comparison to RT-PCR. Our results support a potential role of NanoString's technology as a robust and cost-effective technique that could be used in the detection of fusion transcripts and implemented into the diagnostic algorithm of B-ALL.

Effective and economical column-based method for RNA isolation from animal cells.

OBJECTIVES: To develop an efficient, economical, and low-toxicity method for the extraction of RNA from animal cells to meet a basic requirement of biological research: the isolation of high-quality RNA. RESULTS: Guanidine hydrochloride was used as a lysis buffer and Na-acetate was used as a wash buffer to extract RNA fragments from TM3 Leydig cells and ovarian granulosa cells efficiently. The functionality of the extracted RNA samples was verified through polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (RT-PCR). PCR results showed that the normal DNA column-based method could guarantee RNA integrity and could be used to amplify gene fragments successfully. RT-PCR analysis showed that the RNA samples isolated through the proposed method could be used to detect the expression levels of steroidogenic acute regulatory protein and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA in TM3 Leydig cells under induction by luteinizing hormone. The proposed method could be used to isolate RNA from mammalian cells and provided RNA yields of > 120 ng/5 × 106 cells. This method provided RNA with purities and yields that are sufficient for cDNA synthesis and PCR amplification in gene expression studies. CONCLUSIONS: The proposed RNA extraction method has the advantages of low toxicity, safe handling, and low cost. Isolation can be completed in 20 min. The proposed method can be used to extract RNA from various animal cell samples and is worth promoting.

Performance and workflow assessment of six nucleic acid extraction technologies for use in resource limited settings.

Infectious disease nucleic acid amplification technologies (NAAT) have superior sensitivity, specificity, and rapid time to result compared to traditional microbiological methods. Recovery of concentrated, high quality pathogen nucleic acid (NA) from complex specimen matrices is required for optimal performance of several NA amplification/detection technologies such as polymerase chain reaction (PCR). Fully integrated NAAT platforms that enable rapid sample-to-result workflows with minimal user input are generally restricted to larger reference lab settings, and their complexity and cost are prohibitive to widespread implementation in resource limited settings (RLS). Identification of component technologies for incorporation of reliable and affordable sample preparation with pathogen NA amplification/detection into an integrated platform suitable for RLS, is a necessary first step toward achieving the overarching goal of reducing infectious disease-associated morbidity and mortality globally. In the current study, we evaluate the performance of six novel NA extraction technologies from different developers using blinded panels of stool, sputum and blood spiked with variable amounts of quality-controlled DNA- and/or RNA-based microbes. The extraction efficiencies were semi-quantitatively assessed using validated real-time reverse transcription (RT)-PCR assays specific for each microbe and comparing target-specific RT-PCR results to those obtained with reference NA extraction methods. The technologies were ranked based on overall diagnostic accuracy (analytical sensitivity and specificity). Sample input and output volumes, total processing time, user-required manual steps and cost estimates were also examined for suitability in RLS. Together with the performance analysis, these metrics were used to select the more suitable candidate technologies for further optimization of integrated NA amplification and detection technologies for RLS.

A robust, cost-effective method for DNA, RNA and protein co-extraction from soil, other complex microbiomes and pure cultures.

The soil microbiome is inherently complex with high biological diversity, and spatial heterogeneity typically occurring on the submillimetre scale. To study the microbial ecology of soils, and other microbiomes, biomolecules, that is, nucleic acids and proteins, must be efficiently and reliably co-recovered from the same biological samples. Commercial kits are currently available for the co-extraction of DNA, RNA and proteins but none has been developed for soil samples. We present a new protocol drawing on existing phenol-chloroform-based methods for nucleic acids co-extraction but incorporating targeted precipitation of proteins from the phenol phase. The protocol is cost-effective and robust, and easily implemented using reagents commonly available in laboratories. The method is estimated to be eight times cheaper than using disparate commercial kits for the isolation of DNA and/or RNA, and proteins, from soil. The method is effective, providing good quality biomolecules from a diverse range of soil types, with clay contents varying from 9.5% to 35.1%, which we successfully used for downstream, high-throughput gene sequencing and metaproteomics. Additionally, we demonstrate that the protocol can also be easily implemented for biomolecule co-extraction from other complex microbiome samples, including cattle slurry and microbial communities recovered from anaerobic bioreactors, as well as from Gram-positive and Gram-negative pure cultures.

Proteinase K treatment improves RNA recovery from thyroid cells fixed with liquid-based cytology solution.

OBJECTIVE: Fine-needle aspiration biopsy (FNAB), an important diagnostic tool given its simplicity, safety, and cost-effectiveness, is fast becoming a popular procedure in the diagnosis of thyroid diseases. Generally, cells isolated from biopsies are transferred directly to microscope slides to prepare smears for cytopathological examination; however, the technical difficulties of this procedure often cause poor reproducibility, which limits the accuracy of diagnostic results. Liquid-based cytology (LBC), in which isolated cells are collected in a fixative solution, is advantageous in that it facilitates the preparation of homogenous cytological specimens. However, LBC has not been applied to molecular diagnoses, such as RNA expression-based diagnosis, mainly because of difficulties in cell recovery and RNA isolation. This study was aimed to improve RNA extraction from papillary cancer-derived K1 cells and thyroid FNAB specimens suspended in LBC solutions. RESULTS: K1 cells suspended in CytoRich-Red and CytoRich-Blue, fixatives for LBC, were efficiently recovered by trapping to glass-fiber filters. Importantly, subsequent Proteinase K treatment was essential for efficient RNA extraction from the fixed cells. This finding was also applicable to RNA extraction from CytoRich-Red-fixed thyroid FNAB specimens processed in the same way. Consistently, U6 small nuclear RNA was detected in these RNA samples by reverse transcription-polymerase chain reaction.

Three Methods to Purify Leukocytes and RNA Quality Assessment.

Leukocytes function as central effectors in innate immunity (such as phagocytosis) as well as adaptive immunity (e.g., antigen-dependent T cell activation), and serve as an important resource in the fields of translational medicine, precision medicine, and cell therapy. Isolation of leukocytes from whole blood is necessary for high-quality RNA and downstream research. This process is susceptible to the variability of many factors, such as blood collection, isolation reagents, and extraction methods. In this study, three methods were applied for leukocytes separation, followed by RNA extraction and quality testing to evaluate the methods. Results showed that leukocytes were purified using lymphocyte separation medium (LSM), optimized LSM method, or red blood cell lysis buffer (RBC lysis), and RNA quality met the basic requirements for downstream studies. Although considering the simplicity of the procedure and RNA quality from donated samples, the RBC lysis method should be recommended to biobanks for further research.

A technical assessment of the porcine ejaculated spermatozoa for a sperm-specific RNA-seq analysis.

The study of the boar sperm transcriptome by RNA-seq can provide relevant information on sperm quality and fertility and might contribute to animal breeding strategies. However, the analysis of the spermatozoa RNA is challenging as these cells harbor very low amounts of highly fragmented RNA, and the ejaculates also contain other cell types with larger amounts of non-fragmented RNA. Here, we describe a strategy for a successful boar sperm purification, RNA extraction and RNA-seq library preparation. Using these approaches our objectives were: (i) to evaluate the sperm recovery rate (SRR) after boar spermatozoa purification by density centrifugation using the non-porcine-specific commercial reagent BoviPureTM; (ii) to assess the correlation between SRR and sperm quality characteristics; (iii) to evaluate the relationship between sperm cell RNA load and sperm quality traits and (iv) to compare different library preparation kits for both total RNA-seq (SMARTer Universal Low Input RNA and TruSeq RNA Library Prep kit) and small RNA-seq (NEBNext Small RNA and TailorMix miRNA Sample Prep v2) for high-throughput sequencing. Our results show that pig SRR (~22%) is lower than in other mammalian species and that it is not significantly dependent of the sperm quality parameters analyzed in our study. Moreover, no relationship between the RNA yield per sperm cell and sperm phenotypes was found. We compared a RNA-seq library preparation kit optimized for low amounts of fragmented RNA with a standard kit designed for high amount and quality of input RNA and found that for sperm, a protocol designed to work on low-quality RNA is essential. We also compared two small RNA-seq kits and did not find substantial differences in their performance. We propose the methodological workflow described for the RNA-seq screening of the boar spermatozoa transcriptome. ABBREVIATIONS: FPKM: fragments per kilobase of transcript per million mapped reads; KRT1: keratin 1; miRNA: micro-RNA; miscRNA: miscellaneous RNA; Mt rRNA: mitochondrial ribosomal RNA; Mt tRNA: mitochondrial transference RNA; OAZ3: ornithine decarboxylase antizyme 3; ORT: osmotic resistance test; piRNA: Piwi-interacting RNA; PRM1: protamine 1; PTPRC: protein tyrosine phosphatase receptor type C; rRNA: ribosomal RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; SRR: sperm recovery rate; tRNA: transfer RNA.

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA.

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species. Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family, it could be easily used to other prokaryote and eukaryote species.

Have you tried spermine? A rapid and cost-effective method to eliminate dextran sodium sulfate inhibition of PCR and RT-PCR.

The Dextran Sulfate Sodium (DSS) induced colitis mouse model is commonly used to investigate human inflammatory bowel disease (IBD). Nucleic acid extracts originating from these animals are often contaminated with DSS, which is a strong inhibitor of many enzymatic based molecular biology reactions including PCR and reverse-transcription (RT). Methods for removing DSS from nucleic acids extracts exist for RNA, but no effective protocol for DNA or cDNA is currently available. However, spermine has previously been shown to be an effective agent for counteracting DSS inhibition of polynucleotide kinase, which led to the hypothesis, that spermine could be used to counteract DSS inhibition of PCR and RT. We investigated the means of adding spermine in an adequate concentration to PCR based protocols (including qPCR, two-step RT-qPCR, and amplicon sequencing library preparation) to remove DSS inhibition. Within the range up to 0.01g/L, spermine can be added to PCR/qPCR or RT prophylactically without a significant reduction of reaction efficiency. Addition of spermine at the concentration of 0.08g/L can be used to recover qualitative PCR signal inhibited by DSS in concentrations up to 0.32g/L. For optimal quantitative analysis, the concentration of spermine requires fine adjustment. Hence, we present here a simple fluorometric based method for adjusting the concentration of spermine ensuring an optimal efficiency of the reaction exposed to an unknown concentration of DSS. In conclusion, we demonstrate a cost effective and easy method to counteract DSS inhibition in PCR and two-step RT-qPCR. Fixed or fine-tuned concentrations of spermine can be administered depending on the qualitative or quantitative character of the analysis.

Comparative Analysis of Single-Cell RNA Sequencing Methods.

Single-cell RNA sequencing (scRNA-seq) offers new possibilities to address biological and medical questions. However, systematic comparisons of the performance of diverse scRNA-seq protocols are lacking. We generated data from 583 mouse embryonic stem cells to evaluate six prominent scRNA-seq methods: CEL-seq2, Drop-seq, MARS-seq, SCRB-seq, Smart-seq, and Smart-seq2. While Smart-seq2 detected the most genes per cell and across cells, CEL-seq2, Drop-seq, MARS-seq, and SCRB-seq quantified mRNA levels with less amplification noise due to the use of unique molecular identifiers (UMIs). Power simulations at different sequencing depths showed that Drop-seq is more cost-efficient for transcriptome quantification of large numbers of cells, while MARS-seq, SCRB-seq, and Smart-seq2 are more efficient when analyzing fewer cells. Our quantitative comparison offers the basis for an informed choice among six prominent scRNA-seq methods, and it provides a framework for benchmarking further improvements of scRNA-seq protocols.

Isolation of RNA from milk somatic cells as an alternative to biopsies of mammary tissue for nutrigenomic studies in dairy ewes.

Nutrigenomic studies of mammary lipogenesis in ruminants often rely on the use of mammary tissue (MT) collected either by biopsy or at slaughter. However, isolating RNA from milk would be a useful and cost-effective technique that may avoid distress to the animal and facilitate the collection of samples in time series experiments. This assay was therefore conducted to test the hypothesis that RNA extracted from milk somatic cells (MSC) in dairy sheep would be a feasible alternative to the performance of MT biopsies for nutrigenomic analyses. To meet this objective, 8 lactating Assaf ewes were divided in 2 groups and offered a total mixed ration without supplementation (control) or supplemented with 2.4% dry matter of fish oil, which was known not only to elicit milk fat depression but also to downregulate the expression of some candidate genes involved in mammary lipogenesis. Total RNA was extracted from MSC and biopsied MT to examine whether the potential changes in the abundance of transcripts was similarly detected with both RNA sources. Milk fatty acid profile was also analyzed by gas chromatography, and variations in mRNA abundance were determined by reverse transcription quantitative PCR. Values of RNA integrity number were always ≥7.7. The expected and designed decrease of milk fat concentration with fish oil (-29%), was associated with a lower transcript abundance of genes coding for enzymes involved in fatty acid activation (ACSS1), de novo synthesis (ACACA and FASN), uptake from plasma lipids (LPL), and esterification of fatty acids to glycerol (LPIN1), as well as of a transcription factor that may regulate their expression (INSIG1). Stable mRNA levels were showed in other candidate genes, such as FABP3, GPAT4, or SCD. Changes due to the dietary treatment were similarly detected with both RNA sources (MSC and MT biopsies), which supports the initial hypothesis and would validate the use of milk as an alternative RNA source for nutrigenomic analyses in dairy sheep.

Quality assessment and preservation of RNA from biobank tissue specimens: a systematic review.

It is well recognised that genomic, proteomic and biomarker studies require properly annotated and well-characterised biospecimens. Consequently, this necessitates biobanks to collect, store and distribute biospecimens under stringent quality control and assurance measures. However, despite this realisation, there remains a lack of standardisation in quality management among biobanks and consensus as to which quality indicators provide the optimal molecular diagnostic performance tools and information for biospecimens. In an attempt to identify key factors that predict tissue specimen integrity and quality, this systematic review investigated the measures reported in the literature, which characterised the collection, processing and storage of high-quality tissue specimens. Our findings demonstrated RNA integrity, alone, may not be an effective measure of tissue quality. Furthermore, the frequently reported parameters related to biospecimen integrity, such as storage time, temperature, time to cryopreservation and tissue morphology were also not effective indicators of quality control and assurance. These findings suggest that it is unlikely that a single marker will provide the optimal diagnostic and performance information for biospecimens, but rather, a panel of markers assessing the molecular integrity of the lifespan of the biospecimen is required. Further work is needed to identify which factors predict specimen integrity and quality in biobanked tissue specimens.

Evaluation of a low-cost procedure for sampling, long-term storage, and extraction of RNA from blood for qPCR analyses.

BACKGROUND: In large clinical trials, where RNA cannot be extracted immediately after sampling, preserving RNA in whole blood is a crucial initial step in obtaining robust qPCR data. The current golden standard for RNA preservation is costly and designed for time-consuming column-based RNA-extraction. We investigated the use of lysis buffer for long-term storage of blood samples for qPCR analysis. METHODS: Blood was collected from 13 healthy adults and diluted in MagMAX lysis/binding solution or PAXgene Blood RNA tubes and stored at -20 °C for 0, 1, or 4 months before RNA extraction by the matching method. RNA integrity, yield and purity were evaluated and the methods were compared by subsequent analyses of the gene expression levels of 18S, ACTB, IL1B, IL1RN, IL1R2, and PGK1 using qPCR. RESULTS: The MagMAX system extracted 2.3-2.8 times more RNA per mL blood, with better performance in terms of purity, and with comparable levels of integrity relative to the PAXgene system. Gene expression analysis using qPCR of 18S, ACTB, IL1B, IL1RN, IL1R2, and the promising blood-specific reference gene, PGK1, revealed negligible differences (<1-fold) between the samples stored in MagMAX lysis/binding solution over time and between samples stored and extracted by the two systems. CONCLUSIONS: The MagMAX system can be used for storage of human blood for up to 4 months and is equivalent to the PAXgene system for RNA extraction. It furthermore, provides a means for significant cost reduction in large clinical trials.

Optimizing Frozen Sample Preparation for Laser Microdissection: Assessment of CryoJane Tape-Transfer System®.

Laser microdissection is an invaluable tool in medical research that facilitates collecting specific cell populations for molecular analysis. Diversity of research targets (e.g., cancerous and precancerous lesions in clinical and animal research, cell pellets, rodent embryos, etc.) and varied scientific objectives, however, present challenges toward establishing standard laser microdissection protocols. Sample preparation is crucial for quality RNA, DNA and protein retrieval, where it often determines the feasibility of a laser microdissection project. The majority of microdissection studies in clinical and animal model research are conducted on frozen tissues containing native nucleic acids, unmodified by fixation. However, the variable morphological quality of frozen sections from tissues containing fat, collagen or delicate cell structures can limit or prevent successful harvest of the desired cell population via laser dissection. The CryoJane Tape-Transfer System®, a commercial device that improves cryosectioning outcomes on glass slides has been reported superior for slide preparation and isolation of high quality osteocyte RNA (frozen bone) during laser dissection. Considering the reported advantages of CryoJane for laser dissection on glass slides, we asked whether the system could also work with the plastic membrane slides used by UV laser based microdissection instruments, as these are better suited for collection of larger target areas. In an attempt to optimize laser microdissection slide preparation for tissues of different RNA stability and cryosectioning difficulty, we evaluated the CryoJane system for use with both glass (laser capture microdissection) and membrane (laser cutting microdissection) slides. We have established a sample preparation protocol for glass and membrane slides including manual coating of membrane slides with CryoJane solutions, cryosectioning, slide staining and dissection procedure, lysis and RNA extraction that facilitated efficient dissection and high quality RNA retrieval from CryoJane preparations. CryoJane technology therefore has the potential to facilitate standardization of laser microdissection slide preparation from frozen tissues.

Electroelution of nucleic acids from polyacrylamide gels: a custom-made, agarose-based electroeluter.

Polyacrylamide electrophoresis is routinely used for small-scale preparative and analytical separations. The incomparably high-resolution separations achieved by this technique, however, have not been widely exploited to the large-scale preparative isolation of biological molecules from contaminants, mainly because of difficulties in the recovery of the desired molecule from the gel matrix. Electroelution is an effective procedure applied for this purpose. However, commercially available, high-cost electroeluters are required for achieving high recovery yields. Here, we describe a custom-made electroeluter that combines low-cost, high-recovery yields, short times of electroelution, and convenience in the manipulation of sensitive samples.