Probing Clinical Samples
The use of real-time qPCR to detect and quantify RNA sequences in blood, tissue, or other types of biological samples is complicated by the instability of the RNA and the changes in transcript concentrations that can occur between sample collection and testing due to post-collection gene induction, cell death, and enzymatic degradation.
“A change in the CT [cycle threshold] value of 10–12 in the sample material is not unusual,” said Ralf Wyrich, Ph.D., associate director R&D, diagnostic sample preparation and stabilization at Qiagen. The CT is a representation of fluorescent signal accumulation, with CT values being inversely proportional to the amount of target nucleic acid in a sample.
Dr. Wyrich described a technique for post-collection stabilization using the PreAnalytiX PAXgene blood RNA tube, which preserves the transcript profile of cellular RNA in blood for up to three days at 18–25ºC, for up to five days at 2–8ºC, and for long-term archiving at -20ºC to -70ºC. The PAXgene blood-collection and RNA-stabilization system has received U.S. FDA clearance and CE mark in Europe for use in molecular diagnostic testing based on real-time PCR.
He also presented data demonstrating an approximate 1 log enrichment of miRNA on average from blood collected in a PAXgene Blood RNA tube compared to standard RNA-extraction procedures using a dedicated sample-preparation kit for the isolation of small RNA species.
A second PAXgene product was designed to preserve the RNA in bone- marrow samples, and the newest PAXgene system targets the collection and preservation of tissue samples to allow for both classical histomorphology and PCR-based molecular analysis to be performed on the same specimen.
The commonly used method for tissue preservation relies on formalin fixation, which does not stabilize the RNA content of the sample and, furthermore, preserves the tissue through chemical crosslinking of molecules, which interferes with qPCR analysis. Currently available nucleic acid stabilization technologies for use in tissue samples do not preserve the morphology of the tissue material.
The PAXgene tissue container exposes the sample first to a fixation reagent and then to a stabilization reagent. In this way the same sample can be sectioned for histomorphologic analysis and processed with either a RNA, DNA, or miRNA kit for nucleic acid purification and analysis.
“The lack of standardization in preanalytical sample handling was recognized by the European Commission, which funds several projects in this field,” said Dr. Wyrich. Qiagen is spearheading a European consortium called SPIDIA (standardization and improvement of generic preanalytical tools and procedures for in vitro diagnostics), which is funded by the European Union, with a total four-year budget of €13 million ($18.7 million). The goal of the 16 participating companies and institutions representing 11 countries is to standardize preanalytical procedures and protocols for molecular diagnostic applications.
qPCR is a potent technique for microRNA expression profiling to assess gene regulation, yet the characteristics of miRNAs present challenges for assay design. miRNAs are only 19–22 nucleotides in length, making design of DNA primers without overlap a challenge.
In addition, they have high sequence homology, and miRNAs within a family may differ by only a single nucleotide. They also have a wide range of GC content, from as little as 25% to as much as 98%. Low GC content makes it difficult to design primers with a melting temperature high enough for PCR, whereas a high GC content makes it difficult to avoid background interference from the formation of primer dimers.
By introducing locked nucleic acid (LNA™) bases into both the forward and reverse PCR primers included in its new miRCURY LNA™ Universal RT microRNA PCR system, Exiqon was able to design short primers with high specificity and sensitivity for miRNA analysis.
“LNAs increase sensitivity and help discriminate between mismatches,” said Ditte Andreasen, Ph.D., senior scientist at Exiqon. She presented data showing that including LNAs in PCR primers increases the sensitivity by about 100 times in comparison to using DNA primers. She also showed how LNAs can lead to greater discriminatory power between single nucleotide mismatches by increasing melting temperature between a mismatch and perfect match target.
Exiqon’s new miRNA qPCR platform incorporates the one-tube Universal RT (reverse transcription) reaction for cDNA synthesis, eliminating the need to perform multiple gene-specific reverse transcriptase reactions and preamplification, according to the company. A single Universal RT reaction performed on an RNA sample as small as 40 ng generates enough material for two 384-well plates, allowing for qPCR analysis of 730 different miRNAs.
Dr. Andreasen reported the results of studies using the LNA Universal RT system to profile miRNA expression in clinical samples: formalin-fixed paraffin-embedded tissue samples, as well as blood serum and plasma samples. She demonstrated the ability to quantify miRNA expression levels compared to reference standards, to discriminate single nucleotide differences, and to generate miRNA signatures. She described efforts under way at the company to develop colon cancer diagnostic qPCR assays using the system, assessing disease-related miRNA expression in biopsy samples.