RNAlater Samples Research Articles

Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces

Feces contain intestinal bacteria and exfoliated epithelial cells that may provide useful information concerning gastrointestinal tract health. Intestinal bacteria that synthesize or metabolize potential carcinogens and produce anti-tumorigenic products may have relevance to colorectal cancer, the second most common cause of cancer deaths in the USA. To facilitate epidemiological studies relating bacterial and epithelial cell DNA and RNA markers, preservative/extraction methods suitable for self-collection and shipping of fecal samples at room temperature were tested. Purification and PCR amplification of fecal DNA were compared after preservation of stool samples in RNAlater (R) or Paxgene (P), or after drying over silica gel (S) or on Whatman FTA cards (W). Comparisons were made to samples frozen in liquid nitrogen (N2). DNA purification methods included Whatman (accompanying FTA cards), Mo-Bio Fecal (MB), Qiagen Stool (QS), and others. Extraction methods were compared for amount of DNA extracted, DNA amplifiable in a real-time SYBR-Green quantitative PCR format, and the presence of PCR inhibitors. DNA can be extracted after room temperature storage for five days from W, R, S and P, and from N2 frozen samples. High amounts of total DNA and PCR-amplifiable Bacteroides spp. DNA (34% ± 9% of total DNA) with relatively little PCR inhibition were especially obtained with QS extraction applied to R preserved samples (method QS-R). DNA for human reduced folate carrier (SLC19A1) genomic sequence was also detected in 90% of the QS-R extracts. Thus, fecal DNA is well preserved by methods suitable for self-collection that may be useful in future molecular epidemiological studies of intestinal bacteria and human cancer markers.

Safe and legal shipment of tissue samples: does it affect DNA quality?

Safe and legal shipment of tissue samples: does it affect DNA quality?

Molecular markers to differentiate between pancreatic cancer and normal pancreas/chronic pancreatitis: Gene expression profiling study

21150 Background: The aim of the study was to analyze the gene expression profile of pancreatic cancer by multivariate methods of class prediction. Methods: The snap-frozen or RNA-later preserved samples of 18 pancreatic adenocarcinomas, 9 chronic pancreatitis cases and 6 specimens collected from microscopically unchanged pancreas (N/CP) were analyzed by HG-U133 Plus 2.0 oligonucleotide microarrays (Affymetrix). The obtained dataset was pre-processed using GC-RMA method, gene selection was carried out both by class comparison methods (Welch test with Benjamini-Hochberg correction, False Discovery Rate FDR<5%) and by our own algorithms of class prediction, based on Support Vector Machines technique (Recurrent Feature Replacement and Bootstrap-Based Feature Ranking). Real- time quantitative PCR (Q-PCR) was carried out on Applied Biosystems 7900 HT machine, with Universal Probe Library (Roche) fluorescent probes and normalization by three reference genes index (geNorm, Vandesompele et al.). Results: We compared gene expression profiles between pancreatic cancer samples and N/CP specimens. 23850 probesets significantly differentiated between these three classes (FDR<5%). No ideal discrimination between cancer and N/CP samples was possible by any of single markers. We selected the optimal multi- gene classifier by Support Vector Machines, using Bootstrap-Based Feature Ranking method. The smallest classifier resulting in 100% accuracy consisted of three genes, 45 genes were included in more than half of the diagnostic genesets obtained during bootstrapping process. 14 genes were selected for Q-PCR validation, again none of them ideally discriminated between cancer and normal specimens, with the area under the receiver-operating-characteristic curve ranging from 0.82–0.93. Three-gene combinations allowed for proper classification of all samples. Conclusions: The multi-gene classifier, derived both by microarray technique and Q-PCR analysis, is properly discriminating between pancreatic cancer and chronic pancreatitis/normal pancreas. At least three genes must be included in the classifier to obtain satisfying accuracy. MO and MJ equally contributed to the study. No significant financial relationships to disclose.

Preservation of RNA for functional analysis of separated alleles in yeast: comparison of snap-frozen and RNALater® solid tissue storage methods

The aim of the present study was to compare RNALater with the usual method of liquid nitrogen snap freezing as a surrogate mRNA preservation method for functional analysis of separated alleles in yeast (FASAY). A total of 81 patients with transitional cell carcinoma of the bladder underwent fresh tissue biopsies directly transferred into RNALater and stored at room temperature or at 4 degrees C for increasing time intervals until RNA processing. From this cohort of patients, 53 paired snap-frozen and RNALater preservative-suspended tissues were obtained. Samples immediately frozen in liquid nitrogen were further stored at -80 degrees C. Of the 81 RNALater samples, 14 were not processed for FASAY because of RNA degradation. Of the remaining 67 samples, 15 (22%) were FASAY-positive. Identical FASAY results were found for 50 of 53 (94.4%) paired samples and the percentage of red yeast colonies was highly correlated (Cohen's kappa<0.82; p<0.00001). A single p53 missense mutation was found in each of the three discordant positive FASAY and was identical in each concordant positive sample (10/53). Storing samples in RNALater at room temperature for 3 days and at 4 degrees C for less than 1 month provided high-quality mRNA suitable for FASAY. Our results demonstrate that RNALater is a suitable and flexible alternative to snap freezing for FASAY analysis.

EVALUATION OF METHODS FOR DURATION OF PRESERVATION OF RNA QUALITY IN RAT LIVER USED FOR TRANSCRIPTOME ANALYSIS

In The Toxicogenomics Project, about 150 chemicals are administered to rats, and gene expression in the liver analyzed by Affymetrix GeneChip and stored in the database. As the quality of RNA greatly influences the accuracy of gene expression data, conditions of the storage of the sample are very important. Recently, an RNA stabilization solution, RNAlater, has become commercially available. In this study, the new storage method was compared with the traditional storage method (stored in freezer or liquid nitrogen) under various conditions by looking at the degradation of RNA assessed by its total yield, OD260/280 ratio, 28S/18S ratio, and quantity of beta-actin. It was confirmed that RNAlater preserved the liver tissue sample by maintaining the quality of RNA for one year (in liquid N(2) or -80 degrees C), for 3 days (4 degrees C), or for 2 hr (room temperature) without degradation of RNA. Quality of RNA samples dissolved in buffer RLT and stored at -20 degrees C tended to decrease, but samples stored at -80 degrees C were almost equivalent to those stored in liquid nitrogen. In conclusion, we recommend the following procedure for preservation of liver tissue for extraction of RNA: 1) tissues removed should be put into chilled RNAlater as soon as possible; 2) samples in RNAlater must be stored overnight or longer at 4 degrees C and can be left for as long as 2 weeks without freezing; 3) samples in RNAlater can be stored for at least one year under less than -20 degrees C and 4) samples dissolved in buffer RLT can be preserved at least for one year under -80 degrees C.