CDNA synthesis

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

Total RNA is isolated from the lymphocytes according to standard procedures and used as a template for radioactive labeled cDNA synthesis. The purified cDNA is used as probe for cDNA expression arrays. The advantages of this method as compared to other array systems are as follows (1) Radioactive-labeled probes are more sensitive than fluorescent-labeled probes and therefore need less sample RNA. (2) The primers used in the cDNA synthesis match the genes represented on the array. (3) The primer sequences are longer compared to other array systems, which increases the hybridization fidelity of RNA to the matching correct set of genes and therefore reduces mismatch reactions. 

A spin column from the kit is inserted into a 2-mL collection tube, and the sample is pipeted onto the column. The column is centrifuged at 1 l,000g for 1 min, and the flowthrough is saved to evaluate the cDNA synthesis efficiency. The collection tube is discarded in an appropriate container for radioactive waste. 

To elute the cDNA synthesis probe, the spin column is inserted into a clean 1.5-mL microcentrifuge mbe 100 pL buffer NE are added to the column and soaked into it for 2 min. The column is centrifuged as described in step 9, with the flowthrough now containing the labeled cDNA probe. 

RNA to initiate cDNA synthesis. All cellular mRNA contains multiple repeats of adenine bases (poly-A tails). Therefore the complementary thymine bases (oligo-dT) can be used as a primer that binds to the mRNA template required for the reverse transcriptase to synthesize the cDNA. In the case of pancreatic mRNAs (Figure 4.2), the signihcantly higher mRNA for insulin compared with other proteins allowed success in isolating the insulin-specihc cDNA. Subsequent insertion of cDNA into a bacterial expression vector allowed the production of functional insulin that is now marketed as a successful therapeutic product (Figure 4.2). 

Chen, H. and Gold, L. (1994) Selection of high-affinity RNA ligands to reverse transcriptase inhibition of cDNA synthesis and RNase H activity. Biochemistry, 33, 8746-8756. 

Rinse with 200 pL of IX RT buffer and flick out the remaining liquid before proceeding with the cDNA synthesis step. 

The methods outlined below consist of 1) tube coating with the capture antibody, 2) sample extraction from potato, 3) virus capture in the coated tubes, 4) cDNA synthesis from the captured viruses and 5) PCR amplification of the cDNA. Tube coating requires an overnight incubation, so should be set up on the day prior to extractions. 

Downstream application [PCR (polymerase chain reaction), cloning, labeling, blotting, RT (reverse transcriptase)-PCR, cDNA synthesis, RNAse protection assays, gene therapy, etc.]... 

Purification of mRNA and subsequent single-strand cDNA synthesis by reverse transcription (RT) were achieved all on the same chip [956,957]. 

For first-strand cDNA synthesis SUPERSCRIPT II RT reverse transcriptase (200 U/pL) supplemented with 5X RT buffer (250 mM Tris-HCl, pH 8.3, 375 mM KC1, 15mM MgCl2) (Gibco-BRL), random hexamers (50 ng/pL), 0.1 M DTT, 10 mM dNTP mix, and DEPC-treated water. These solutions should be prepared as RNase-free. We utilize solutions supplemented with cDNA synthesis kit (Gibco-BRL)... 

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