Ice recrystallization inhibitors enable efficient cryopreservation of induced pluripotent stem cells: A functional and transcriptomic analysis.

The successful use of human induced pluripotent stem cells (iPSCs) for research or clinical applications requires the development of robust, efficient, and reproducible cryopreservation protocols. After cryopreservation, the survival rate of iPSCs is suboptimal and cell line-dependent. We assessed the use of ice recrystallization inhibitors (IRIs) for cryopreservation of human iPSCs. A toxicity screening study was performed to assess specific small-molecule carbohydrate-based IRIs and concentrations for further evaluation. Then, a cryopreservation study compared the cryoprotective efficiency of 15 mM IRIs in 5 % or 10 % DMSO-containing solutions and with CryoStor® CS10. Three iPSC lines were cryopreserved as single-cell suspensions in the cryopreservation solutions and post-thaw characteristics, including pluripotency and differential gene expression were assessed. We demonstrate the fitness-for-purpose of 15 mM IRI in 5 % DMSO as an efficient cryoprotective solution for iPSCs in terms of post-thaw recovery, viability, pluripotency, and transcriptomic changes. This mRNA sequencing dataset has the potential to be used for molecular mechanism analysis relating to cryopreservation. Use of IRIs can reduce DMSO concentrations and its associated toxicities, thereby improving the utility, effectiveness, and efficiency of cryopreservation.

Implementing routine monitoring for nuclease contamination of equipment and consumables into the quality Management system of a laboratory.

Nucleases are ubiquitous in the environment, present in biospecimens and widely used in many laboratory processes. However, in the wrong context, as contaminants, they have catastrophic potential because of their ability to rapidly degrade nucleic acids whilst retaining high resilience to inactivation. Although laboratories undertake rigorous precautions to prevent nuclease contamination, such measures are not infallible. In 2015, we devised and integrated a novel routine nuclease testing regimen into our Quality Management System that uses cleavable, fluorescent DNA and RNA substrates to detect, monitor and control for nuclease contamination in our laboratory processes, equipment and consumables. The testing regimen enables us to identify higher-risk activities, design our laboratory workflows such that risk is minimized and help fulfil our obligations in respect of ISO 20387:2018 General Requirements for Biobanking and ISO 17025 Testing and Calibrations Laboratory standards, both of which stipulate that environmental conditions in our laboratory must be monitored with defined quality control criteria. In seventeen rounds of testing (30 Test Items per round), 1.1 % of RNase tests and 0.2 % of DNase tests returned elevated nuclease levels (≥2.90 x 10-9 U RNase or 1.67 x 10-3 U DNase) and we were able to take remedial action. In no instance was an elevated nuclease level consequential in terms of an impact on sample quality. We present our protocols, results and observations.

Improving Yields in Multi-analyte Extractions by Utilizing Post-homogenized Tissue Debris.

In multi-analyte extractions, tissue is typically homogenized in a lysis buffer, and then DNA, RNA, and protein are purified from the supernatant. However, yields are typically lower than in dedicated, single-analyte extractions. In a two-part experiment, we assessed whether yields could be improved by revisiting the normally discarded, post-homogenized tissue debris. We initially performed additional homogenizations, each followed by a simultaneous extraction. These yielded no additional RNA, 13% additional DNA (which became progressively more degraded), and 161.7% additional protein (which changed in proteome when analyzed using SDS-PAGE). We then digested post-homogenized tissue debris from a simultaneous extraction using proteinase K and extracted DNA using silica spin columns or alcohol precipitation. An average additional DNA yield of 27.1% (silica spin columns) or 203.9% (alcohol precipitation) was obtained with/without compromising DNA integrity (assessment by long-range PCR, DNA Integrity Numbers, and size at peak fluorescence of electropherogram). Validation using a cohort of 65 tissue blocks returned an average additional DNA yield of 31.6% (silica columns) and 54.8% (alcohol precipitation). Users can therefore refreeze the homogenized remnants of tissue blocks rather than disposing of them and then perform additional DNA extractions if yields in the initial multi-analyte extractions were low.

Do Tissues Fixed in a Non-crosslinking Fixative Require a Dedicated Formalin-free Processor?

We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]+ve), a formalin-free system (NBF-ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF-ve, RNA from NBF+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9-2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF+ve and NBF-ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem.

Why Formalin-fixed, Paraffin-embedded Biospecimens Must Be Used in Genomic Medicine: An Evidence-based Review and Conclusion.

Fresh-frozen tissue is the "gold standard" biospecimen type for next-generation sequencing (NGS). However, collecting frozen tissue is usually not feasible because clinical workflows deliver formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Some clinicians and researchers are reticent to embrace the use of FFPE tissue for NGS because FFPE tissue can yield low quantities of degraded DNA, containing formalin-induced mutations. We describe the process by which formalin-induced deamination can lead to artifactual cytosine (C) to thymine (T) and guanine (G) to adenine (A) (C:G > T:A) mutation calls and perform a literature review of 17 publications that compare NGS data from patient-matched fresh-frozen and FFPE tissue blocks. We conclude that although it is indeed true that sequencing data from FFPE tissue can be poorer than those from frozen tissue, any differences occur at an inconsequential magnitude, and FFPE biospecimens can be used in genomic medicine with confidence.

Effect of Different Proteinase K Digest Protocols and Deparaffinization Methods on Yield and Integrity of DNA Extracted From Formalin-fixed, Paraffin-embedded Tissue.

DNA extracted from formalin-fixed, paraffin-embedded tissue sections is often inadequate for sequencing, due to poor yield or degradation. We optimized the proteinase K digest by testing increased volume of enzyme and increased digest length from the manufacturer's protocol using 54 biospecimens, performing the digest in centrifuge tubes. Doubling the quantity of proteinase K resulted in a median increase in yield of 96%. Applying the optimized proteinase K protocol to sections deparaffinized on microscope slides generated a further increase in yield of 41%, but only at >50,000 epithelial tumor cells/section. DNA yield now correlated with (χ2 = 0.84) and could be predicted from the epithelial tumor cell number. DNA integrity was assayed using end point multiplex PCR (amplicons of 100-400 bp visualized on a gel), quantitative PCR (qPCR; Illumina FFPE QC Assay), and nanoelectrophoresis (DNA Integrity Numbers [DINs]). Generally, increases in yield were accompanied by increases in integrity, but sometimes qPCR and DIN results were conflicting. Amplicons of 400 bp were almost universally obtained. The process of optimization enabled us to reduce the percentage of samples that failed published quality control thresholds for determining amenability to whole genome sequencing from 33% to 7%.

Does vacuum centrifugal concentration reduce yield or quality of nucleic acids extracted from FFPE biospecimens?

Vacuum centrifugal (SpeedVac) concentration is commonly applied to nucleic acids extracted from formalin-fixed paraffin-embedded (FFPE) sections, but with an unknown impact. We concentrated DNA and RNA from FFPE biospecimens using different time/temperature SpeedVac combinations of up to 30 min concentration at 45 °C, then used spectrophotometry, spectrofluorometry, RIN, PERM, DV200, qRT-PCR, DIN and the Illumina FFPE QC Assay to assess the changes in quantity, purity and integrity induced by the concentration process. We found the effects of SpeedVac concentration to be inconsequential, but an aliquot of elution buffer should be concentrated for use as the blank in spectrophotometry assays.

Cold Ischemia Score: An mRNA Assay for the Detection of Extended Cold Ischemia in Formalin-Fixed, Paraffin-Embedded Tissue.

Although there are thousands of formalin-fixed paraffin-embedded (FFPE) tissue blocks potentially available for scientific research, many are of questionable quality, partly due to unknown preanalytical variables. We analyzed FFPE tissue biospecimens as part of the National Cancer Institute (NCI) Biospecimen Preanalytical Variables program to identify mRNA markers denoting cold ischemic time. The mRNA was extracted from colon, kidney, and ovary cancer FFPE blocks (40 patients, 10-12 hr fixation time) with 1, 2, 3, and 12 hr cold ischemic times, then analyzed using qRT-PCR for 23 genes selected following a literature search. No genes tested could determine short ischemic times (1-3 hr). However, a combination of three unstable genes normalized to a more stable gene could generate a "Cold Ischemia Score" that could distinguish 1 to 3 hr cold ischemia from 12 hr cold ischemia with 62% sensitivity and 84% specificity.

Extracting DNA from FFPE Tissue Biospecimens Using User-Friendly Automated Technology: Is There an Impact on Yield or Quality?

DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue blocks is amenable to analytical techniques, including sequencing. DNA extraction protocols are typically long and complex, often involving an overnight proteinase K digest. Automated platforms that shorten and simplify the process are therefore an attractive proposition for users wanting a faster turn-around or to process large numbers of biospecimens. It is, however, unclear whether automated extraction systems return poorer DNA yields or quality than manual extractions performed by experienced technicians. We extracted DNA from 42 FFPE clinical tissue biospecimens using the QiaCube (Qiagen) and ExScale (ExScale Biospecimen Solutions) automated platforms, comparing DNA yields and integrities with those from manual extractions. The QIAamp DNA FFPE Spin Column Kit was used for manual and QiaCube DNA extractions and the ExScale extractions were performed using two of the manufacturer's magnetic bead kits: one extracting DNA only and the other simultaneously extracting DNA and RNA. In all automated extraction methods, DNA yields and integrities (assayed using DNA Integrity Numbers from a 4200 TapeStation and the qPCR-based Illumina FFPE QC Assay) were poorer than in the manual method, with the QiaCube system performing better than the ExScale system. However, ExScale was fastest, offered the highest reproducibility when extracting DNA only, and required the least intervention or technician experience. Thus, the extraction methods have different strengths and weaknesses, would appeal to different users with different requirements, and therefore, we cannot recommend one method over another.

RNA and microRNA Stability in PAXgene-Fixed Paraffin-Embedded Tissue Blocks After Seven Years' Storage.

OBJECTIVES: To evaluate the stability of RNA and microRNA (miRNA) in PAXgene-fixed paraffin-embedded tissue blocks after 7 years' storage. METHODS: RNA and miRNA were extracted from PAXgene-fixed paraffin-embedded (PFPE) blocks in 2009 then stored at -80°C. Seven years later, RNA and miRNA were again extracted from the same blocks. RNA and miRNA integrity in the 2009 and 2016 extractions were compared using RNA integrity number (RIN), paraffin-embedded RNA metric (PERM), reverse transcription polymerase chain reaction (RT-PCR) for different amplicon lengths, and quantitative RT-PCR (qRT-PCR) for three mRNA and three miRNA targets. RESULTS: In PFPE blocks, mRNA was poorer in 2016 extractions compared to the 2009 extractions in all blocks and all assays applied, with transcripts degrading at different rates in the same blocks. For miRNA, qRT-PCR showed no statistically significant differences between 2009 and 2016 extractions. CONCLUSIONS: mRNA in PFPE tissue blocks degrades at room temperature storage over 7 years.

A Critical Evaluation of the PAXgene Tissue Fixation System: Morphology, Immunohistochemistry, Molecular Biology, and Proteomics.

OBJECTIVES: To evaluate the PAXgene tissue fixation system. METHODS: Clinical biospecimens (n = 46) were divided into PAXgene-fixed paraffin-embedded (PFPE), formalin-fixed paraffin-embedded (FFPE), and fresh-frozen (FF) blocks. PFPE and FFPE sections were compared for histology (H&E staining) and immunohistochemistry (14 antibodies) using tissue microarrays. PFPE, FFPE, and FF samples were compared in terms of RNA quality (RNA integrity number, polymerase chain reaction [PCR] amplicon length, and quantitative reverse transcription PCR), DNA quality (gel electrophoresis and methylation profiling) and protein quality (liquid chromatography-mass spectrometry [LC-MS/MS]). RESULTS: PFPE protocol optimization was required in most cases and is described. RNA extracted from PFPE sections was considerably less degraded than that from FFPE sections but more degraded than that from FF blocks. Genomic-length DNA was extracted from PFPE and FF biospecimens, and methylation profiling showed PFPE and FF biospecimens to be almost indistinguishable. Only degraded DNA was extracted from FFPE biospecimens. PFPE sections yielded peptides that were slightly less amenable to LC-MS/MS analysis than FFPE sections, but FF gave slightly better results. CONCLUSIONS: While it cannot be envisaged that PAXgene will replace formalin in a routine clinical setting, for specific projects or immunodiagnostics involving biospecimens destined for immunohistochemical or histologic staining and DNA or RNA analyses, PAXgene is a viable option.

The effect of long-term -80°C storage of thyroid biospecimens on RNA quality and ensuring fitness for purpose.

AIMS: To establish whether RNA degrades in long-term storage at -80°C and whether RNA integrity numbers (RINs) determine 'fitness for purpose' in severely degraded RNA. METHODS: RNA was extracted from 549 thyroid biospecimens stored at -80°C for 0.1-10.9 years then their RINs correlated with storage time. RT-PCR for 65, 265, 534 and 942 base pair amplicons of hydroxymethylbilane synthase was used to measure amplicon length in RNA from cryopreserved and FFPE biospecimens that were equally degraded according to RIN. RESULTS: Storage time did not correlate with RIN. Longer amplicons were obtained from cryopreserved samples than FFPE samples with equal RINs. CONCLUSIONS: RNA does not degrade in thyroid biospecimens stored for long periods of time at -80°C. Although RINs are known to predict amenability to analytical platforms in good quality samples, this prediction is unreliable in severely degraded samples.

An Independent Evaluation of the CryoXtract Instruments' CXT350 Frozen Sample Aliquotter Using Tissue and Fecal Biospecimens.

The ability to take targeted multiple cores from a single frozen biospecimen would enable several research projects to be fueled from one biospecimen, a small piece of tissue to be quality-control tested, and for pathologically-discrete areas of a biospecimen (e.g., tumor, stromal, and normal tissue) to be selectively sampled for comparative analyses. CryoXtract Instruments' CXT350 Frozen Sample Aliquotter can potentially achieve this by producing multiple cores from one cryopreserved biospecimen without thawing either the parent biospecimen or its daughter cores. It therefore has the potential to add significant value to a tissue banking workflow. We have evaluated its performance while using 614 cores from fecal, liver, kidney, lung, heart, and colon biospecimens. Coring densities of up to five complete and four fragmentary cores per cm(3) are achievable using 3 mm coring probes. Median core weights for tissue were 14.1-17.2 mg (depending on tissue type) and cores ≤325 mg could be taken from fecal biospecimens (depending on the fill-depth of the tube). The coefficient of variation for multiple cores taken from a fecal biospecimen was 11.7%. Between-sample contamination did not occur. RNA Integrity numbers and qRT-PCR analysis demonstrated that coring induced a statistically significant impact on RNA quality that was inconsequential in magnitude and in our view does not represent a barrier for the effective utilization of the technology.

How Severely Is DNA Quantification Hampered by RNA Co-extraction?

The optional RNase digest that is part of many DNA extraction protocols is often omitted, either because RNase is not provided in the kit or because users do not want to risk contaminating their laboratory. Consequently, co-eluting RNA can become a "contaminant" of unknown magnitude in a DNA extraction. We extracted DNA from liver, lung, kidney, and heart tissues and established that 28-52% of the "DNA" as assessed by spectrophotometry is actually RNA (depending on tissue type). Including an RNase digest in the extraction protocol reduced 260:280 purity ratios. Co-eluting RNA drives an overestimation of DNA yield when quantification is carried out using OD 260 nm spectrophotometry, or becomes an unquantified contaminant when spectrofluorometry is used for DNA quantification. This situation is potentially incompatible with the best practice guidelines for biobanks issued by organizations such as the International Society for Biological and Environmental Repositories, which state that biospecimens should be accurately characterized in terms of their identity, purity, concentration, and integrity. Consequently, we conclude that an RNase digest must be included in DNA extractions if pure DNA is required. We also discuss the implications of unquantified RNA contamination in DNA samples in the context of laboratory accreditation schemes.

Replacing ß-mercaptoethanol in RNA extractions.

RNA extractions are potentially compromised in terms of both yield and quality by ribonucleases (RNases). The pungent and toxic reducing agent ß-mercaptoethanol (ß-ME), therefore, is commonly added to the biospecimen's lysis buffer to aid in RNase deactivation. Using different tissue types (liver tissue, kidney tissue, and cell pellets), extraction kits (RNeasy Mini Kit, Illustra RNA Spin Mini Kit, and PureLink Mini Kit), RNA quality assays (RNA integrity numbers [RINs] and quantitative real-time polymerase chain reaction [qRT-PCR]), yield assessments, and in vitro functional RNase assays (RNaseAlert Kit), we demonstrate that ß-ME should be replaced by the less toxic dithiothreitol (DTT) alternative.

DNA fingerprinting: a quality control case study for human biospecimen authentication.

This case study illustrates the usefulness of the DNA fingerprinting method in biobank quality control (QC) procedures and emphasizes the need for detailed and accurate record keeping during processing of biological samples. It also underlines the value of independent third-party assessment to identify points at which errors are most likely to have occurred when unexpected results are obtained from biospecimens.

Simultaneously extracting DNA, RNA, and protein using kits: is sample quantity or quality prejudiced?

Interdisciplinary "omics" research and the stringent quality requirements of array-based technologies require the simultaneous yet efficient extraction of DNA, RNA, and protein from the same tissue block. However, the few commercially available simultaneous extraction kits have not been evaluated. We compare the TriplePrep (GE Healthcare) and AllPrep (Qiagen) kits using good, intermediate, and poor quality tissue with specialist single-extract methods: Puregene (DNA), RNeasy (RNA), and homogenizations into buffer (protein). The following parameters were evaluated: DNA-yield (total DNA and double-stranded), purity (260:280 and 260:230), and integrity (gel electrophoresis); RNA-yield, purity, and integrity (RNA integrity numbers [RINs] and quantitative reverse transcription polymerase chain reaction [Q-RT-PCR]); protein-yield and quality (two-dimensional difference gel electrophoresis [2D-DIGE]). Puregene DNA yields were 183% and 506% those of TriplePrep and AllPrep, respectively. For RNA, AllPrep and RNeasy were indistinguishable, but their yields were 412% to 588% those of TriplePrep (depending on block condition) and their between-sample variability was better. TriplePrep protein yields were 57% those of the control, and 6.9% of the gel spots were more than 2-fold altered. However, AllPrep yields were 20% of the control, with 11% of the gel spots being more than 2-fold altered. Therefore, TriplePrep outperformed AllPrep in DNA and protein extractions, the reverse was true for RNA, but neither kit achieved optimal efficiency because both yield and quality were compromised.

Insights into blood feeding by schistosomes from a proteomic analysis of worm vomitus.

Whilst the schistosome tegument has been intensively studied there is little information about processes in the gut, the other major interface with the bloodstream, apart from the well characterised cascade of proteases involved in haemoglobin digestion. To gain insights into gut function we undertook a proteomic analysis of worm vomitus and performed in vitro erythrocyte feeding experiments. Additional to known gut constituents we identified two proline carboxypeptidases as well as enzymes capable of hydrolysing carbohydrate and ester linkages. Schistosome serpin and a2 macroglobulin protease inhibitors were also present. A series of "carrier proteins", principally lipid-binding saposins and cholesterol-binding NPC-2 were also detected, together with ferritins and calumenin that bind ferric iron and calcium, respectively. The presence of these lysosomal proteins and other lysosomal markers in the vomitus, plus observations on the cytology of the gut epithelium suggest that lysosomes directly secrete their contents into the gut lumen to digest incoming plasma constituents as well as haemoglobin. It is also likely that the carrier proteins function to sequester essential organic and inorganic nutrients for uptake into the epithelium. The feeding experiments indicate that erythrocytes are uncoated as they pass through the oesophagus, intersecting with its secretions, whilst the endocytosis of space-filling dextran into the gut epithelium provides a potential mechanism for carrier uptake by macropinocytosis.

Antimicrobials and in vitro systems: antibiotics and antimycotics alter the proteome of MCF-7 cells in culture.

Cell culture is widely used to study gene or protein changes in response to experimental conditions. The value of such experiments depends on stringent control and understanding of the in vitro environment. Despite well-documented evidence describing toxic effects in the clinical setting, antibiotics and antimycotics are routinely used in cell culture without regard for their potential toxicity. We cultured MCF-7 breast cancer cells in the presence/absence of antibiotics (penicillin/streptomycin) and/or the antimycotic amphotericin B. Differential protein expression was assessed using 2D-DIGE and MALDI-MS/MS. Antibiotics caused 8/488 spots (1.3% of the protein) to be generally down-regulated. The affected proteins were principally chaperones and cytoskeletal. In marked contrast, amphotericin B induced a more dramatic response, with 33/488 spots (9.5% of the total protein) generally up-regulated. The proteins were mostly involved in chaperoning and protein turnover. Combining antibiotics and amphotericin B had little overall effect, with only one (unidentified) protein being up-regulated. As this study identifies differential protein expression attributable to antibiotics/antimycotics, we urge caution when comparing and interpreting proteomic results from different laboratories where antibiotics/antimycotics have been used. We conclude that as antibiotics and antimycotics alter the proteome of cultured cells in markedly different ways their use should be avoided where possible.