Notes: This study identified two novel transcript variants of the estrogen receptor ERβ that were expressed in the ERα-negative breast cancer cell line MDA-MD-231. These variants were identified after amplification of ERβ transcripts from the breast cancer cell line by RT-PCR. The amplification products were then excised from gels and subcloned into the pTARGET™ Mammalian Expression Vector prior to sequencing. COS1 cells, which do not express the estrogen receptor, were then stably transfected with full-length ERβ or one of the splice variants and the effects on cell proliferation, apoptosis, and estrogen response were evaluated. In COS1 cells expressing either ERβ or the transcript variants cell proliferation decreased and basal apoptosis (caspase 3/7 activity) increased, compared to cells transfected with vector alone. Exposure to therapeutic doses of tamoxifen induced apoptosis in cells expressing the full-length ERβ but not in cells expressing either of the variant isoforms. (3618)

Notes: The authors investigated the effect of abasic RNA on DNA primer extension by various reverse transcriptase, including those from HIV-1, avian myeloblastosis virus (AMV) and Moloney muring leukemia virus (MMLV) and determined the preference of dNTP incorporation at abasic sites. The authors found that trans-lesion synthesis readily occurs with HIV-1 and to a lesser extent AMV RT, but MMLV RT aborts synthesis. The MMLV used in this study was M-MLV Reverse Transcriptase, RNAse H Minus, from Promega. (3909)

Notes: To examine why there are two splicing systems present in multicellular organisms, the authors used morpholino oligomers complementary to the branch-site recognition elements of U2 or U12 small nuclear RNA to specifically suppress the splicing function in EL4 and Jurkat cells. After transfection with the morpholino oligos, the cells were harvested, and total RNA was isolated followed by DNase-treatment. In a 20µl reverse transcription reaction, 0.8µg total RNA was reverse transcribed with 100ng oligo(dT)₁₅ primer and 200 units of M-MLV Reverse Transcriptase, RNase H Minus. PCR was then performed using 5µl of the diluted (1:10) reverse transcriptase reaction with 2.5 units GoTaq^® DNA polymerase in a 50µl reaction for 28–31 cycles. (3278)

Notes: The SV Total RNA Isolation System was used to isolate total RNA from Alteromonas cultures. Five micrograms of the total RNA was used in reverse transcription reactions with M-MLV Reverse Transcriptase, RNase H Minus. cDNA chiB (chitinase B) products from the reaction were quantified by real-time PCR with SYBR green dye. (3023)

Notes: M-MLV Reverse Transcriptase, RNase H Minus, was used to make first-strand cDNA from total and mRNA samples isolated from Arabidopsis seedlings and cell culture. cDNA was then diluted 1:20 in real-time PCR to quantitatively assess GORK, PP2CA, AKT2/3, KIN2 transcript levels. Data was normalized to actin transcript levels.  (3022)

Notes: The SV Total RNA Isolation System was used to isolate total RNA from primary human coronary artery endothelial cells. The isolated RNA was used to make gene array ³³P-labeled targets on nylon cDNA microarray filters using Oligo(dT) and M-MLV Reverse Transcriptase, RNase H-. The same material was used for first-strand synthesis in RT-PCR. (2697)

Notes: Poly A+ RNA was isolated directly from PBS-perfused mouse lungs with the PolyATtract^® System 1000. The isolate poly A+ RNA was used for quantitative two-step RT-PCR using M-MLV RNase Hminus Reverse Transcriptase as well as RNasin^® Ribonuclease inhibitor for first step cDNA synthesis. (0821)