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Biospecimen RNA Quality Control in Reverse-Transcription, Quantitative PCR (RT-qPCR) Clinical Tests

Stanoszek, Lauren M.
http://rave.ohiolink.edu/etdc/view?acc_num=mco1430323793

Abstract Details

2015, Doctor of Philosophy (PhD), University of Toledo, Biomedical Sciences (Cancer Biology).
Although numerous platforms exist to measure gene expression, RT-qPCR assays remain the most sensitive and accurate. The accuracy and reproducibility of any RT-qPCR –based molecular test is dependent on adherence to stringent quality control criteria involving biospecimen collection and storage, RNA isolation and reverse transcription and PCR amplification of cDNA. The following work was focused on three parameters of RNA quality control: RNA integrity, reverse transcription efficiency and PCR efficiency in three RT-qPCR-based assays. All studies used a form of competitive, RT-qPCR known as standardized, reverse transcription PCR (StaRT-PCR) that allows measurement of each gene relative to a known number of internal standards in each PCR assay using the same PCR primers, thus controlling for PCR efficiency. Additionally all studies use a novel Reverse Transcription Standards Mixture (RTSM), prepared by combining known amounts of in-vitro transcribed, synthetic External RNA Control Consortium (ERCC) RNA standards to determine the efficiency of each RT reaction including a specific volume of RTSM. The first study was designed to gather empiric data to determine the impact of room temperature storage time, different RT primers and RNA input in the RT reaction on BCR-ABL1 and ß-glucuronidase (GUSB) cDNA yield in both whole blood samples from patients with chronic myelogenous leukemia and anonymized normal whole blood mixed with BCR-ABL1-positive K562 cells. After completing these experiments, we concluded that the hypothesis that BCR-ABL1/GUSB measurement in blood samples can be reliably measured within 48 hours is not supported by the results. Further the hypothesis that the current standard random priming method for reverse transcription in BCR-ABL1 testing is suboptimal was supported by our data. Therefore to achieve accurate and sensitive BCR-ABL1 normalized to GUSB measurement, whole blood should be processed within 24 hours after collection and gene-specific primers should be used in the RT reaction. The second study tested the hypothesis that increasing RNA input in the RT reaction of RNA extracted from a smallmouth bass infected with Viral Hemorrhagic Septicemia virus (VHSv) strain IVb would significantly change the measurement of VHSv-IVb – N gene and reference genes actb1 and ef1a compared to a baseline RNA input due to reductions in RT efficiency measured by the RTSM. While certain results supported the hypothesis that increasing amounts of fish RNA in RT reactions reduces RT efficiency, thus causing significant changes in VHSv-IVb/actb1 measurement across different RNA inputs, actb1/ef1a values did not statistically change with increasing RNA input. Therefore, a standard RNA input per RT reaction should be employed during VHSv measurement, for which 1µg total RNA/90 µL reaction is recommended. The third study tested two hypotheses. First, we hypothesized that certain RNA extraction methods specific for formalin-fixed, paraffin-embedded (FFPE) samples would provide RNA eluates from A549 FFPE samples with different levels of RT inhibitors that would reduce the RT efficiency of ERCC 171 RNA within the RTSM. Second, we hypothesized that use of gene-specific primers in RT reactions of RNA extracted from surgical FFPE lung tissue would yield a higher quantity of ACTB cDNA compared to random hexamers. The results supported the hypotheses, demonstrating that the RNeasy® FFPE kit provides less RT inhibitors co-extracted with RNA, and ACTB cDNA yield increased with the use of gene-specific RT priming. The work presented here will demonstrate the significance of RNA quality control criteria to ensure the accuracy and sensitivity of three RT-qPCR clinical tests.
James Willey, MD (Advisor)
Keith Crist, PhD (Committee Member)
David Dignam, PhD (Committee Member)
Neocles Leontis, PhD (Committee Member)
Kenneth Muldrew, MD (Committee Member)
David Weaver, PhD (Committee Member)
311 p.
Molecular Biology

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Citations

  • Stanoszek, L. M. (2015). Biospecimen RNA Quality Control in Reverse-Transcription, Quantitative PCR (RT-qPCR) Clinical Tests [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco1430323793

    APA Style (7th edition)

  • Stanoszek, Lauren. Biospecimen RNA Quality Control in Reverse-Transcription, Quantitative PCR (RT-qPCR) Clinical Tests . 2015. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco1430323793.

    MLA Style (8th edition)

  • Stanoszek, Lauren. "Biospecimen RNA Quality Control in Reverse-Transcription, Quantitative PCR (RT-qPCR) Clinical Tests ." Doctoral dissertation, University of Toledo, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=mco1430323793

    Chicago Manual of Style (17th edition)

mco1430323793
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© 2015, all rights reserved.
This open access ETD is published by University of Toledo Health Science Campus and OhioLINK.