Quantitative real-time RT-PCR (qRT-PCR) of zebrafish transcripts: Optimization of RNA extraction, quality control considerations, and data analysis

Cold Spring Harbor Protocols

Volumn 4, Issue 10, 2009, Pages

  Lan, Chuan Ching ^a   Tang, Rongying ^a   Leong, Ivone Un San ^a   Love, Donald R ^a,b  

^a UNIVERSITY OF AUCKLAND   (New Zealand)

^b AUCKLAND CITY HOSPITAL   (New Zealand)

Author keywords

[No Author keywords available]

Indexed keywords

RNA;

ANALYTICAL EQUIPMENT; ARTICLE; DATA ANALYSIS; EMBRYO; FISH GENETICS; NONHUMAN; QUALITY CONTROL; QUANTITATIVE ANALYSIS; REAL TIME POLYMERASE CHAIN REACTION; REVERSE TRANSCRIPTION; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; RNA ANALYSIS; RNA EXTRACTION; RNA ISOLATION; RNA PURIFICATION;

DANIO RERIO;

EID: 70949108110     PISSN: None     EISSN: 15596095     Source Type: Journal    
DOI: 10.1101/pdb.prot5314     Document Type: Article

References (18)

1. Andersen CL, Jensen JL, Ørntoft TF. 2004. Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64: 5245-5250.
2. Apte A, Daniel S. 2009. PCR primer design. Cold Spring Harb Protoc doi: 10.1101/pdb.ip65.
3. Barbas CF III, Burton DR, Scott JK, Silverman GJ. 2007. Quantitation of DNA and RNA. Cold Spring Harb Protoc doi: 10.1101/pdb.ip47.
4. Kusser W, Javorschi S, Gleeson MA. 2006. Real-time RT-PCR: cDNA synthesis. Cold Spring Harb Protoc doi: 10.1101/pdb.prot4114.
5. Liu W, Saint DA. 2002a. A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal Biochem 302: 52-59.
6. Liu W, Saint DA. 2002b. Validation of a quantitative method for real time PCR kinetics. Biochem Biophys Res Commun 294: 347-353.
7. -ΔΔCT method. Methods 25: 402-408.
8. Malnati MS, Scarlatti G, Gatto F, Salvatori F, Cassina G, Rutigliano T, Volpi R, Lusso P. 2008. A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load. Nat Protoc 3: 1240-1248.
9. Morimoto M, Morimoto M, Whitmire J, Star RA, Urban JF Jr, Gause WC. 2006. cDNA synthesis and real-time PCR using RNA from laser-captured cells. Cold Spring Harb Protoc doi: 10.1101/pdb.prot4108.
10. Pfaffl MW. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45. doi: 10.1093/ nar/29.9.e45.
11. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using pair-wise correlations. Biotechnol Lett 26: 509-515.
12. Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM. 2003. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339: 62-66.
13. Roux KH. 2009. Optimization and troubleshooting in PCR. Cold Spring Harb Protoc doi: 10.1101/pdb.ip66.
14. Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, Moorman AFM. 2009. Amplification efficiency: Linking baseline and bias in the analysis of quantitative PCR data. Nucl Acids Res 37: e45. doi: 10.1093/nar/gkp045.
15. Sambrook J, Russell DW. 2006. Amplification of cDNA generated by reverse transcription of mRNA. Cold Spring Harb Protoc doi: 10.1101/ pdb.prot3837.
16. Tang R, Dodd A, Lai D, McNabb WC, Love DR. 2007. Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization. Acta Biochim Biophys Sin (Shanghai) 39: 384-390.
17. Tichopad A, Didier A, Pfaffl MW. 2004. Inhibition of real-time RT-PCR quantification due to tissue-specific contaminants. Mol Cell Probes 18: 45-50.
18. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: research0034.1-research0034.11.