| qRT-PCR to detect RNA present at low levels |
| Analyzing viral RNA, lncRNA, and other challenging RNA molecules |
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| qRT-PCR是检测低拷贝RNA的一种比较实用的技术。qRT-PCR的应用包括在感染性疾病研究中定量病毒RNA，分析非编码RNA如miRNA或lncRNA，研究临床样品如甲醛固定、石蜡包埋（FFPE）样品的检测。低拷贝数的转录物，如编码转录因子的mRNA，是某些细胞和组织类型具有固有的RNA水平，通常会限制基因表达分析。此外，一些转录本，如编码转录因子的mRNA在非常低的水平上表达，需要qRT-PCR技术的灵敏度和精密度来检测。 |
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| 然而，反转录酶活性的效率可能是qRT-PCR敏感性的主要限制因素。在这里，我们提供关于qRT-PCR的部分文献资料，包括病毒RNA、lncRNA、miRNA、冷冻或FFPE临床样品和低拷贝mRNA的研究应用例。 |
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| PrimeScript Reverse Transcriptase是Takara公司依据M-MLV (Moloney Murine Leukemia Virus)来源的RTase开发的一种反转录酶。Primescript的灵敏度和特异性有大量的文献支持。比如，该酶在total RNA pool中可以检测到10-21 mol miRNA，另外，该酶可以特异性检出含有单碱基错配的miRNA分化(Yao, B. et al. (2009) RNA 15:1787–1794)。下表列出PrimeScript RTase的试剂盒用途。 |
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| PrimeScript kit selection guide |
| 制品名称 |
Code No. |
推荐用途 |
| PrimeScript RT Master Mix (Perfect Real Time) |
RR036A |
1st strand cDNA synthesis for two-step qRT-PCR. Master mix includes primers and RTase. |
| PrimeScript RT Reagent Kit (Perfect Real Time) |
RR037A |
cDNA synthesis for two-step qRT-PCR. Primers and RTase provided as separate components. |
| PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) |
RR047A |
Removal of genomic DNA contamination during qRT-PCR. |
| One Step PrimeScript RT-PCR Kit (Perfect Real Time) |
RR064A |
One-step qRT-PCR with 5' nuclease-based assays. (Probe not included) |
| One-Step TB Green PrimeScript RT-PCR Kit II (Perfect Real Time) |
RR086A |
One-step qRT-PCR with TB Green detection(High amplification efficiency and specificity). |
| One-Step TB Green PrimeScript RT-PCR Kit (Perfect Real Time) |
RR066A |
One-step qRT-PCR with TB Green detection. |
| One-Step TB Green PrimeScript RT-PCR Kit PLUS (Perfect Real Time) |
RR096A |
One-step qRT-PCR with TB Green detection(Effective inhibition of nonspecific amplification). |
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| 使用例：使用PrimeScript检测低拷贝RNA |
| 由于样本或细胞类型的不同或固有的低表达，目的RNA可能拷贝数较低。例如，某些组织（如肝脏）每毫克组织有微克量的RNA，但是其骨或脂肪在同等量的组织中只有1%的RNA。另外，样品本身是一个限制因素。如冷冻或FFPE组织，它可能是不可替代的，那么通过它获得可靠的qRT-PCR数据显得至关重要。 |
| 此外，在长期储存过程中也可能会造成RNA损失。调节蛋白如转录因子对细胞功能有很强的影响，但也可能在低水平表达。还有一些RNA分子如非编码RNA（前miRNA、miRNA、lncRNA等）也可能以低拷贝数存在。使用传统技术检测这些目的RNA可能是比较困难的。下面介绍一些使用PrimeScript的文献。仅供参考。 |
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| Viral RNA |
| TB Green PrimeScript RT-PCR Kit用于检测小鼠肺组织中的甲型流感病毒: 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 7, e34865 (2012). |
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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). |
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| Long non-coding RNA (lncRNA) |
| PrimeScript RT Master Mix (Perfect Real Time) 用于分析lncRNA: Mizutani, R., et al. Identification and characterization of novel genotoxic stress-inducible nuclear long noncoding RNAs in mammalian cells. PLoS ONE 7, e34949 (2012). |
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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. |
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| miRNA and pre-miRNA |
| PrimeScript Reverse Transcriptase 用于检测10-21mol miRNA: Yao, B., et al Quantitative analysis of zeptomole microRNAs based on isothermal ramification amplification. RNA 15, 1787 (2009). |
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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. |
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| PrimeScript RT Reagent Kit用于分析pre-miRNAs: Tang, Y., et al. Genome-wide analysis reveals diversity of rice intronic miRNAs in sequence structure, biogenesis and function. PLoS ONE 8, e63938 (2013). |
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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. |
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| Frozen or FFPE samples |
| PrimeScript RT Reagent Kit用于分析人子宫肌层与子宫平滑肌瘤(frozen and FFPE samples): Makino, K., et al. Inhibition of uterine sarcoma cell growth through suppression of endogenous tyrosine kinase B signaling. PLoS ONE7, e41049 (2012). |
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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. |
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| Low copy number mRNA: Transcription factors |
| PrimeScript Reverse Transcriptase用于分析转录因子的表达: Yamamoto, M., et al. Shared and distinct functions of the transcription factors IRF4 and IRF8 in myeloid cell development. PLoS ONE 6, e25812 (2011). |
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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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