MRNA quantification

Pfaffl MW, Hageleit M (2001) Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR. Biotech Lett 23 275-282... 

In order to test the first hypothesis, we tried to quantify the level of expression of both desaturase genes in Canton-S and Tai. The conventional methods for mRNA quantification such as northern blotting could not be used, as both genes show cross-hybridization over the entire open reading frame. Therefore, we chose to use an adaptation of PCR for accurate quantification of desat mRNA this technique has the advantage of high sensitivity in the study of low-abundance mRNAs and has already been used for desatl quantification (Ueyama et al., 2005). We developed the same technique for desat2. 

Wang T, Brown MJ (1999) mRNA quantification by real time TaqMan polymerase chain reaction validation and comparison with RNase protection. Anal Biochem 269 198-201... 

Results from gene array experiments should be verified by other mRNA quantification methods such as Northern blots3,4, ribonuclease protection assays8,36,128 dot and slot blots72115, or quantitative real time PCR (Q RT-PCR)58. 

Bi WL, Keller-McGandy C, Standaert DG, Augood SJ (2002) Identification of nitric oxide synthase neurons for laser capture microdissection and mRNA quantification. Biotechniques 33 1274-1283... 

Gene expression mRNA quantification and transcriptome analysis... 

Stahlberg, A., Hakansson, J., Xian, X., Semb, H., and Kubista, M., Properties of the reverse transcription reaction in mRNA quantification, Clin. Chem., 50, 509-515,2004. 

Auboeuf D, Rieusset J, Fajas L, Vallier P, Frering V, Riou JP, et al. Tissue distribution and quantification of the expression of mRNAs of peroxisome proliferator-activated receptors and liver X receptor-alpha in humans no alteration in adipose tissue of obese and NIDDM patients. Diabetes 1997 46 1319-1327. 

Depending upon the experimental design, the purpose of the experiment, and the nature of the mRNA template used to program the translational systems, different types of product quantification should (or could) be used. Aside from the quantification of the luciferase synthesized, which is carried... 

RNA Extraction and quantification Indicates total biological structure expensive equipment determined in sofisticated laboratory with specialized equipment quantifying mRNA is still in its developmental stage Lleo et al. (2001)... 

Harris, D.W., Kenrick, M.K., Pither, R.J., Anson, J.G., and Jones, D.A., Development of a high-volume in situ mRNA hybridization assay for the quantification of gene expression utilizing scintillating microplates, Anal. Biochem., 243, 249,1996. 

Quantitative RT PCR (qRT PCR) can be used to accurately determine the levels of messages within given preparations of RNA. qRT PCR thermocyclers provide rapid online detection and quantification of mRNA, however, the initial purchase cost and the cost of reagents may be prohibitive for some laboratories. Methods of semiquantitative RT PCR have been used and good descriptions of these techniques are available (Samhrook and Russell, 2001). However, the same cDNA populations should not be used for differential display reactions and verification that a potential differential display band represents a differentially expressed gene. For this reason, independent cDNA samples should be prepared if both the screening and verification methods rely on PCR. qRT PCR, therefore, should be used in conjunction with other methods to verify that a differential display band represents a differentially expressed gene. 

Sun, A., Ge, J., Siffert, W., and Frey, U. H. (2005) Quantification of allele-specific G-protein beta3 subunit mRNA transcripts in different human cells and tissues by Pyrosequencing. Eur. J. Hum. Genet. 13, 361-369. 

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