The isolated RNA was then treated with audio class DNase I to get rid of contaminating genomic DNA. First strand cDNA was reverse transcribed from 2 _g of total RNA using a Synthesis System package from Invitrogen Corporation. The exact same levels of cDNA were eventually used for the PCR amplification using a SuperPak DNA polymerase kit from Sigma. The rat specific E2F 1 PCR primers were used to generate a fragment of 8-6 bp, Bax PCR primers were used to generate a fragment of 15-0 CTEP bp, and DP5 PCR primers were used to generate a of 80 bp. The expression of actin mRNA was used as a typical. Actin primers were used to create a product of 200 bp; these primers were made to cross a large expanse of intronic sequence between exons 2 and 3 of the rat gene. The nonreactivity of the primers with contaminating genomic DNA was tested by the introduction of the reverse transcriptase enzyme that was omitted by controls from the cDNA synthesis reaction. The PCR services and products were separated on the 14 days agarose gel. Mitochondrion The possible lack of primer dimerization o-r non unique PCR merchandise bands was also examined. Relative gene expression was quantified by realtime quantitative PCR utilizing the ABI PRISM 7700 Sequence Detection System. Real-time PCR was performed employing a SYBR Green PCR system. As SYBR Green binds nonspecifically to each doublestrand DNA product formed throughout amplification, thus, primer concentration and PCR melting temperature were adjusted in order to avoid non-specific PCR services and products. The optimum temperature is whatever provides maximum reading for the specific product once the non specific product can no longer be detected. Quantitative PCR was carried out using the next thermal cycling program, after the optimum temperature have been identified. Stage 1 was undertaken at 95 C for 1-5 min. Phase 2 contained three steps: 9-5 C for 1-5 s; 60 C for 30 s; and 72 C for 30 s. Phase 2 was repeated 40 times. The relative mRNA angiogenesis therapy expression was determined by the standard curve method. In short, actin and E2F 1, Bax o-r Dp5 gene amplifications were run in split up tubes. Standard curves were obtained for many genes through the use of decreasing amounts of cDNA template. PCR reactions were performed in duplicate for standard curves, while samples were analyzed in triplicate, at a final volume of 2-5 di-no source in every cases. For each cDNA format, the cycle threshold required to identify the amplified product was established and semilogarithmic standard plots were drawn. The cDNA levels in the samples were interpolated from the semilogarithmic standard plots and normalized for the cDNA concentration of the control gene, actin. Nonreactivity of the primers was tried from the addition of controls that omitted the cDNA template.

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