Notes: This paper describes a method for detection of polyA RNA in permeablized cells and cell sections. The method is based on incorporation of 5-bromo-2´-deoxyuridine (BrdUTP) into cDNA by AMV reverse transcriptase. The BrdUTP was easily detectable in DNA-RNA duplexes, and undetectable in DNA-DNA duplexes, making it possible to use the method to detect RNA in situ. RNasin® Ribonuclease Inhibitor was used in the reverse transcriptase reaction mix. (4226)

Notes: The authors investigated the effect of neonatal bisphenol A (BPA) exposure in rats on expression of estrogen receptor α (ERα) transcripts. Alternative ERα transcripts in preoptic area of treated and untreated rats were quantified using real-time RT-PCR. Reverse transcription was performed using 4µg of total RNA, 200pmol random primers and 300 units M-MLV Reverse Transcriptase. Real-time PCR was performed using SYBR^® Green I to quantify amplified products. To determine if the changes in BPA-induced ERα transcript expression were caused by DNA methylation, the methylation status of the five ERα promoters was examined by bisulfite modification. Genomic DNA was isolated from rat tissue using the Wizard^® Genomic DNA Purification Kit, denatured with NaOH, then treated with hydroquinone and sodium bisulfite. Prior to methylation-specific PCR, DNA was cleaned up using the Wizard^® DNA Purification Resin as directed by the manufacturer. PCR products were cleaned up again using the Wizard^® SV Gel and PCR Clean-Up System, then subjected to restriction enzyme digestion and agarose gel electrophoresis to reveal methylation-dependent sequence differences. (3911)

Notes: The PolyATtract^® System 1000 was used to isolate polyA+ RNA from Pisium sp. plant tissue. Many details of the isolation are given in the paper. The isolated polyA+ RNA was used for RT-PCR and Northern blot analyses. (0736)