3 race system kit

5′ RACE was performed with gene-specific primers for the sense and antisense transcripts (Supplementary Table 2) using Invitrogen 5′ RACE System and RNA from PC3 and LNCaP cells. cDNA was purified, tailed with dCTP and amplified consecutively with gene specific primers and either Abridged Anchor primer or Abridged Universal Amplification primer provided in the 5′RACE system kit. For 3′RACE, nuclear and chromatin-associated RNA was polyadenylated with Poly(A) tailing kit (Applied Biosystem). Polyadenylated RNA was then retro-transcribed and amplified consecutively with gene-specific primers using Invitrogen 3′ RACE system kit. Final PCR products were cloned into pGEM-T Easy vector (Promega) and sequenced.

RACE was performed with the 5′RACE System and 3′RACE System Kit (Invitrogen, USA) according to the manufacturer’s instructions. Briefly, the 5′RACE System is a set of prequalified reagents intended for synthesis of the first-strand cDNA, purification of the first-strand cDNA, homopolymeric tailing, and preparation of target cDNA for subsequent amplification by PCR. The 3′RACE procedure is also summarized as cDNA synthesis, RNA template degraded with RNase H, and amplificated by PCR. The primers used are as follows (GSP: Gene specific Primer):
5’RACE: cDNA GSP: TAAACAGGTGAAACACT;
PCR GSP: ACATTCGCAAGAGGGTGACAGT;
Nest PCR GSP: CGTCCCCAGGGCACCAGAATA;
3’RACE: GSP1: CAGGACCAGCCAGCCCTTTC;
GSP2: ATGGGAGCCTGGCCTTTGAG;
Primer-887 (1):
F: CACAGCAGCCTCCTCTTAAAC; R: CTTTTCTCTCCCATGCTGAGC
Primer-887 (2):
F: GGCCTTTGAGATTCCTGCGA; R: ATGCCTCAGTCGAAGGGAGA
Primer-887S: F: CTGCTCAGACACCGTTGC; R: GATGTGGTCTCACTCTGTTGC;
Primer-887L: F: CTGCTCAGACACCGTTGC; R: CTTGATGCTTTTACAGGCTCTC