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qRT-PCR to detect RNA present at low levels
Analyzing viral RNA, lncRNA, and other challenging RNA molecules
RT-PCR can be a particularly useful technique for detecting RNA present at low levels. Examples of applications for qRT-PCR include quantifying viral RNA during infectious disease research, analyzing non-coding RNAs such as miRNA or long non-coding RNA (lncRNA) molecules, studying clinical or archival samples such as formaldehyde-fixed, paraffin-embedded (FFPE) samples, and detecting low copy number transcripts such as mRNAs encoding transcription factors. Certain cell and tissue types have inherently low levels of RNA, which ordinarily constrains gene expression analysis efforts. Additionally, some transcripts such as mRNAs encoding transcription factors are expressed at very low levels, requiring the sensitivity and precision of qRT-PCR for detection.
The efficiency of reverse transcriptase enzyme activity, though, can be a limiting factor for the sensitivity of qRT-PCR. Here we present information about PrimeScript kits for qRT-PCR and examples of using PrimeScript RTase for qRT-PCR with viral RNA, lncRNA, miRNA, frozen or FFPE clinical samples, and low copy number mRNA.
Using PrimeScript reverse transcriptase for qRT-PCR to detect low abundance RNA
PrimeScript reverse transcriptase is a highly sensitive and specific RNase H minus recombinant RTase derived from Moloney Murine Leukemia Virus (MMLV). The sensitivity of PrimeScript RTase is well-documented; for example, the enzyme allows detection of zeptomole amounts of miRNA, even in pools of total RNA, while its specificity allows differentiation of miRNAs containing single base mismatches (Yao, B. et al. (2009) RNA 15:1787–1794). Several kits for qRT-PCR using PrimeScript RTase are available, as described in the table below:
RNA molecules of interest may be present at low copy number due to either inherent qualities of samples or cell types, or inherently low expression levels. For example, whereas some tissues (such as liver) have microgram quantities of RNA per mg tissue, others such as bone or adipose may have < 1% as much RNA in an equivalent amount of tissue. In other cases the sample itself is a limiting factor. Archival samples such as frozen or FFPE tissue may be irreplaceable, making it critical to obtain reliable qRT-PCR data.
Additionally, loss of RNA during long-term storage can be problematic. Regulatory proteins such as transcription factors may be expressed at low levels despite their powerful effect on cell function, and some RNA molecules such as noncoding RNA (pre-miRNA, miRNA, lncRNA, etc.) also may be present at low copy number. Detecting such targets may be challenging using conventional techniques. Several articles from the peer-reviewed literature citing use of PrimeScript Reverse Transcriptase for qRT-PCR are summarized below.
Zou, Q., et al. Use of praziquantel as an adjuvant enhances protection and Tc-17 responses to killed H5N1 virus vaccine in mice. PLoS ONE e34865 (2012).
Avian A/H5N1 Influenza A virus is highly pathogenic, and more information is needed about ways to induce broad cytotoxic T lymphocyte responses to killed H5N1 vaccine. The authors reported that the adjuvant Praziquantel (PZQ) boosted protection against a lethal-dose challenge with H5N1 virus in mice. Viral load in mouse lung tissue was assessed by qRT-PCR using One-Step TB Green PrimeScript RT-PCR Kit II (Perfect Real Time).
Mizutani, R., et al. Identification and characterization of novel genotoxic stress-inducible nuclear long noncoding RNAs in mammalian cells. PLoS ONE e34949 (2012).
The authors used bioinformatics methods to identify putative novel lncRNA molecules involved in genotoxic stress response, and PrimeScript RT Master Mix (Perfect Real Time) was used to investigate expression patterns by qRT-PCR.
Yao, B., et al Quantitative analysis of zeptomole microRNAs based on isothermal ramification amplification. RNA 1787 (2009).
The authors report development of a sensitive and specific isothermal ramification amplification (RAM) real-time assay for quantitative analysis of miRNA, allowing accurate quantification of miRNAs in total RNA samples without further enrichment. PrimeScript Reverse Transcriptase was used for the initial reverse transcription of miRNAs to cDNA.
Tang, Y., et al. Genome-wide analysis reveals diversity of rice intronic miRNAs in sequence structure, biogenesis and function. PLoS ONE e63938 (2013).
While most miRNAs are located in intergenic regions, some are present in introns (in-miRNA). The authors identified novel in-miRNA molecules in rice, and used PrimeScript RT Reagent Kit for analysis of pre-miRNA molecules by endpoint RT-PCR.
Makino, K., et al. Inhibition of uterine sarcoma cell growth through suppression of endogenous tyrosine kinase B signaling. PLoS ONE e41049 (2012).
To better understand the regulation of uterine leiomyosarcoma—an aggressive tumor resistant to many treatment regimens—the authors assessed the expression of brain-derived neurotrophic factor (BDNF) and receptor tyrosine kinase B (TrkB) and its ligands in clinical samples of uterine tissues. PrimeScript RT Reagent Kit was used for qRT-PCR analysis of expression levels of TrkB and its ligands were compared among frozen samples of uterine myometrium and leiomyoma and FFPE samples of leiomyosarcoma.
Yamamoto, M., et al. Shared and distinct functions of the transcription factors IRF4 and IRF8 in myeloid cell development. PLoS ONE e25812 (2011).
Interferon regulatory factor (IRF) 8 and IRF4 are hematopoietic cell-specific transcription factors that control the differentiation of dendritic cells and B cells, although the role of IRF8 is more well understood than IRF4. Using PrimeScript Reverse Transcriptase for qRT-PCR, the authors studied the expression patterns of IRF4 and IRF8 themselves, as well as several macrophage-related genes previously known to be regulated by IRF8.
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