| qRT-PCR to detect RNA present at low levels |
| Analyzing viral RNA, lncRNA, and other challenging RNA molecules |
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| qRT-PCR是檢測低拷貝RNA的一種比較實用的技術。qRT-PCR的應用包括在感染性疾病研究中定量病毒RNA，分析非編碼RNA如miRNA或lncRNA，研究臨床樣品如甲醛固定、石蠟包埋（FFPE）樣品的檢測。低拷貝數(shù)的轉錄物，如編碼轉錄因子的mRNA，是某些細胞和組織類型具有固有的RNA水平，通常會限制基因表達分析。此外，一些轉錄本，如編碼轉錄因子的mRNA在非常低的水平上表達，需要qRT-PCR技術的靈敏度和精密度來檢測。 |
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| 然而，反轉錄酶活性的效率可能是qRT-PCR敏感性的主要限制因素。在這里，我們提供關于qRT-PCR的部分文獻資料，包括病毒RNA、lncRNA、miRNA、冷凍或FFPE臨床樣品和低拷貝mRNA的研究應用例。 |
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| PrimeScript Reverse Transcriptase是Takara公司依據(jù)M-MLV (Moloney Murine Leukemia Virus)來源的RTase開發(fā)的一種反轉錄酶。Primescript的靈敏度和特異性有大量的文獻支持。比如，該酶在total RNA pool中可以檢測到10-21 mol miRNA，另外，該酶可以特異性檢出含有單堿基錯配的miRNA分化(Yao, B. et al. (2009) RNA 15:1787–1794)。下表列出PrimeScript RTase的試劑盒用途。 |
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| PrimeScript kit selection guide |
| 制品名稱 |
Code No. |
推薦用途 |
| 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檢測低拷貝RNA |
| 由于樣本或細胞類型的不同或固有的低表達，目的RNA可能拷貝數(shù)較低。例如，某些組織（如肝臟）每毫克組織有微克量的RNA，但是其骨或脂肪在同等量的組織中只有1%的RNA。另外，樣品本身是一個限制因素。如冷凍或FFPE組織，它可能是不可替代的，那么通過它獲得可靠的qRT-PCR數(shù)據(jù)顯得至關重要。 |
| 此外，在長期儲存過程中也可能會造成RNA損失。調(diào)節(jié)蛋白如轉錄因子對細胞功能有很強的影響，但也可能在低水平表達。還有一些RNA分子如非編碼RNA（前miRNA、miRNA、lncRNA等）也可能以低拷貝數(shù)存在。使用傳統(tǒng)技術檢測這些目的RNA可能是比較困難的。下面介紹一些使用PrimeScript的文獻。僅供參考。 |
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| Viral RNA |
| TB Green PrimeScript RT-PCR Kit用于檢測小鼠肺組織中的甲型流感病毒: 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 用于檢測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用于分析人子宮肌層與子宮平滑肌瘤(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用于分析轉錄因子的表達: 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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