Flow cell primers were then extended for 75 sequencing cycles, ultimately yielding clusters of clonally amplified cDNA templates.
To identify homologous recombinants, we established a long range PCR strategy to detect homologous recombinant ES cells (primer sets P1 and P2, Figure 1A).
We observed increased H3K4me3, a hallmark of active transcription, in the 5′ region of the short PDE2A transcript in NHBE cells (primer 2 in Fig. 2D and E).
Since ERa exon6 is also subject to alternative splicing, ERa exon6 as well as ERa standard (primers located in exon1) PCR assays were used as ERa transcript level control for differently transfected cells (primer sequences: Additional file 1: Table S1).
IF staining was used to identify MTDH expression site and cell structure, and western blotting was used for evaluating protein level of EMT markers in cells (Primer sequences for real-time PCR are shown in Supplementary Table S3).
To identify what are the DCPS transcripts present in the patients' cells, primer sets were designed to distinguish isoforms 1, 2 and 3 (Fig. 2A and D and Supplementary Material, Table S2).
PCR was performed on cDNAs from CLL cells (stimulated CLL 31: primers RG657/RG658), MEC1 cells (primers RG659/RG660), and MEC1AID-SRC cells (primers RG661/RG662; sequences of PCR primers listed in Supporting Information Table 2).
While RT PCR is a basic method used for the detection and quantification of RNA expression in cells, primer-independent cDNA synthesis can interfere with RT specificity, and may lead to misinterpretation of the results.
While RT PCR is a basic method used for the detection and quantification of RNA expression in cells, primer-independent cDNA synthesis can interfere with RT specificity, and may lead to misinterpretation of the results, especially when analyzing bidirectional RNA.
