Our protocol consists of a random primed cDNA synthesis step followed by 2 successive rounds of PCR (Figure 1) (17). The first amplification reaction occurs in presence of forward (F) and reverse (R) primers, whereas in the second round, two primers internal to the first amplification product (FN and RN, N for nested) are used to increase specificity. Primer sequences are located in different exons to avoid amplification of genomic DNA and are all designed to anneal at 55°C (first round) or 60°C (second round), so that different cDNAs can be amplified together in a single multiplex experiment. Primers are usually 22 to 24 nucleotides long with a guanosine and cytosine content of 50% to 60%. 5′ and 3′ end of the primers are not complementary to prevent the formation of hairpins or primer dimers. The amplified fragment must be kept short, preferably in the range of 150 to 400 bp. Each couple of primers is first tested in a RTPCR experiment from 100 ng of retina total RNA. These amplifications are carried out for 40 cycles at the appropriate annealing temperature. The RT-PCR products are digested with suitable restriction enzymes to confirm specificity. RNAdependency of the amplification is established by omitting RT during first-strand cDNA synthesis.
Neurons of interest, bystander cells and controls (supernatant and first-strand mixture in absence of a cell) are examined simultaneously in each experiment. Furthermore, six is the highest number of cells that are analyzed at any given time to minimize contamination. The repertory of PCR products should be the same when the number of cycles is increased from 28 + 30 cycles to 40 + 35 cycles. However, the number of cycles must be kept as low as possible to avoid contamination and increase in number of nonspecific signals. The number of cycles required, however, depends on a variety of factors, but especially the abundance of the amplified transcript. In most cases, a single round of 40 cycles is sufficient. Many different RT-PCR strategies are reported in the literature. The first-strand synthesis can be directed by oligo(dT) primers, random examers or antisense primers that recognize specific target sequences. The cDNA thus obtained can then be amplified with specific primers for a single-round PCR or with two-round PCR, where the first amplification occurs in presence of degenerated oligonucleotides and the second with primers specific for the various transcripts. Several methods of detection have been employed, including ethidium bromide staining, Southern blotting, and direct autoradiography of the amplified bands through incorporation of a radioactive nucleotide or primer in the PCR.
3.6 μL of 5× first-strand buffer (KCl 375 mM, MgCl2 15 mM, Tris-HCl 250 mM, pH 8.3); 36 U RNAGUARD; 4 ng Pd(N)6, 1 mM each dNTPs (from Amersham Pharmacia Biotech), 4 mM DTT, 200 U Reverse Transcriptase SUPERSCRIPT II H- (both from Life Technologies).
mRNA, first-round F primer was 5′- CTGGCCTTCCGTGTGTTTCAGTG- 3′, which hybridizes with TH cDNA at nt 915 (GenBank® Accession No. M69200), and the corresponding R primer was 5′-CCGGCTGGTAGGTTTGATCTTGG- 3′, which hybridizes with TH cDNA at nt 1296. The first-round RT-PCR product was 382 bp long. The second-round FN primer was 5′-AGTGCACACAGTACATCCGTCAT- 3′, which hybridizes with TH cDNA at nt 934, and the corresponding RN primer was 5′-GCTGGTAGGTTTGATCTTGGTA- 3′, which hybridizes with TH cDNA at nt 1293. The final nested RT-PCR product was 360 bp long and contained a unique SacI site.
10 μL of 10× RT-PCR buffer (KCl 350 mM, MgCl2 9 mM, Tris-HCl 100 mM, pH 8.3), 40 pM each F and R primers, 1 U Taq DNA polymerase (Roche Molecular Biochemicals).
contains 5 μL of 10× PCR buffer (KCl 500 mM, MgCl2 15 mM, Tris-HCl 100 mM, pH 8.3), 200 μM each dNTPs, 20 pM each FN and RN primers, 1 U Taq DNA polymerase.
Valencia, CA, USA).
Carlsbad, CA, USA).
First-Strand cDNA Synthesis 1. Tubes containing single cells are incubated for 1 minute 30 seconds at 65°C. 2. After cooling in ice for 2 minutes, 18 μL of First Strand Reaction Mixture is added to each tube, and first-strand cDNA is synthesized at 42°C for 1 hour. At the end of the reaction, tubes are cooled on ice. Two Rounds PCR Here, we describe the procedure that we used for amplification of TH mRNA. 1. Eighty microliters of the first-round PCR mixture are added to the tubes. First-round amplification comprises: 2 minutes 30 seconds at 94°C; 10 cycles each consisting of 30 seconds at 94°C, 30 seconds at 55°C, and 1 minute at 72°C; 18 cycles each consisting of 30 seconds at 94°C, 30 seconds at 55°C, and 1 minute at 72°C, with a 5 second extension time added to the final step of each cycle beginning from the second cycle; 10 minutes at 72°C. 2. One microliter of the first-round PCR products is used as a template for 49 μL of the second-round PCR mixture solution. Amplification is carried out as following: 2 minutes 30 seconds at 94°C; 10 cycles each consisting of 30 seconds at 94°C, 30 seconds at 60°C, and 1 minute at 72°C; 20 cycles each consisting of 30 seconds at 94°C, 30 seconds at 60°C, and 1 minute at 72°C, with a 5 second extension time added to the final step of each cycle beginning from the second cycle. Analysis of the Results 1. Ten microliters of the second-round PCR products are analyzed by electrophoresis in 1% Seakem® GTG® and 1% NuSieve® GTG agarose (FMC BioProducts, Rockland, ME, USA), containing 0.5 μg/mL ethidium bromide. 2. The remaining PCR products are purified and desalted using the Qiagen kit. DNA is then digested with SacI to check for specificity. 3. When the experiments at the single-cell level are completed, a single-cell RTPCR product is cloned into the pCR-II vector by using the Original TA Cloning Kit. Both strands are sequenced using the dideoxy-chain termination method. DNA similarities are examined by matching the query sequence to database entries using the basic local alignment search tool (BLAST) algorithm. We rigorously avoided cloning the PCR fragments before the end of the single-cell RT-PCR experiments, because the presence of a recombinant sequence in the laboratory increases the possibility of contamination.
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