Improved microRNA quantification in total RNA from clinical samples

microRNAs are small regulatory RNAs that are currently emerging as new biomarkers for cancer and other diseases. In order for biomarkers to be useful in clinical settings, they should be accurately and reliably detected in clinical samples such as formalin fixed paraffin embedded (FFPE) sections and blood serum or plasma. These types of samples represent a challenge in terms of microRNA quantification. A newly developed method for microRNA qPCR using Locked Nucleic Acid (LNA?)-enhanced primers enables accurate and reproducible quantification of microRNAs in scarce clinical samples. Here we show that LNA?-based microRNA qPCR enables biomarker screening using very low amounts of total RNA from FFPE samples and the results are compared to microarray analysis data. We also present evidence that the addition of a small carrier RNA prior to total RNA extraction, improves microRNA quantification in blood plasma and laser capture microdissected (LCM) sections of FFPE samples.     

Identification of methods for use of formalin-fixed, paraffin-embedded tissue samples in RNA expression profiling

Formalin-fixed paraffin-embedded (FFPE) tissue samples are a potentially valuable resource of expression information for medical research, but are under-utilized due to degradation and modification of the RNA. Using a random primer-based RNA amplification strategy, we have evaluated multiple protocols for the extraction and isolation of RNA from FFPE samples. We found that the RecoverAll RNA isolation procedure with three or four slices (ten-microns in thickness), supplemented with additional DNAse, gave optimal results. RNA integrity as assessed by Agilent Bioanalyzer, and amplification of the 28S ribosomal RNA, were predictive for the number of genes detected on Affymetrix arrays. We obtained expression data for colon and lung tumor and normal FFPE samples and matched frozen samples and found a high correlation between frozen and matched FFPE samples (R² between 0.82 and 0.89), while the signature sets in tumor versus normal comparisons were also quite similar. QPCR confirmed all 16 of the differential expression results from the microarrays that we tested. Differentially expressed signature genes from tumor versus matched normal FFPE tissue from colon and lung were identified as cancer-related, with 95 colon tumor and 67 lung tumor genes identified, respectively.     

Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens

Background

Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens.

Principal Findings

For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses.

Significance

We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies.     

RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain

microRNAs (miRNAs) are small (approximately 22 nucleotide) regulatory RNAs which play fundamental roles in many biological processes. Recent studies have shown that the expression of many miRNAs is altered in various human tumors and some miRNAs may function as oncogenes or tumor suppressor genes. However, with the exception of glioblastoma multiforme, the expression of miRNAs in brain tumors is unknown. Furthermore, methods to profile miRNAs from formalin-fixed, paraffin-embedded (FFPE) archival tissues or to study their cellular and subcellular localization in FFPE tissues have been lacking. Here we report the coordinated miRNA expression analysis from the tissue level to the subcellular level, using the RAKE (RNA-primed, array-based, Klenow Enzyme) miRNA microarray platform in conjunction with Locked Nucleic Acid (LNA)-based in situ hybridization (LNA-ISH) on archival FFPE human brains and oligodendroglial tumors. The ability to profile miRNAs from archival tissues at the tissue level, by RAKE microarrays, and at the cellular level by LNA-ISH, will accelerate studies of miRNAs in human diseases.     

Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues

Archival formalin-fixed paraffin-embedded (FFPE) human tissue collections are typically in poor states of storage across the developing world. With advances in biomolecular techniques, these extraordinary and virtually untapped resources have become an essential part of retrospective epidemiological studies. To successfully use such tissues in genomic studies, scientists require high nucleic acid yields and purity. In spite of the increasing number of FFPE tissue kits available, few studies have analyzed their applicability in recovering high-quality nucleic acids from archived human autopsy samples. Here we provide a study involving 10 major extraction methods used to isolate total nucleic acid from FFPE tissues ranging in age from 3 to 13 years. Although all 10 methods recovered quantifiable amounts of DNA, only 6 recovered quantifiable RNA, varying considerably and generally yielding lower DNA concentrations. Overall, we show quantitatively that TrimGen’s WaxFree method and our in-house phenol-chloroform extraction method recovered the highest yields of amplifiable DNA, with considerable polymerase chain reaction (PCR) inhibition, whereas Ambion’s RecoverAll method recovered the most amplifiable RNA.     

Improved RNA quality and TaqMan<Superscript>®</Superscript>Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials

Background  

Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp) designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62–164 bp).     

A cut-off based approach for gene expression analysis of formalin-fixed and paraffin-embedded tissue samples

Microarray analysis of formalin-fixed and paraffin-embedded (FFPE) tissue seems to be of importance for the detection of molecular marker sets in prostate cancer (PC). The compromised RNA integrity of FFPE tissue results in a high degree of variability at the probe level of microarray data as shown by degradation plot. We tested methods that reduce the variability by including all probes within 300 nucleotides, within 600 nucleotides, or up to a calculated breakpoint with reference to the 3'-end. Accepted PC pathways such as the Wnt signaling pathway could be observed to be significantly regulated within FFPE microarray datasets. The best representation of PC gene expression, as well as better comparability to meta-analysis and fresh-frozen microarray data, could be obtained with a 600-nucleotide cutoff. Beyond the specific impact for PC microarray data analysis we propose a cutoff of 600 nucleotides for samples for which the integrity of the RNA cannot be guaranteed.