RNA ampification University of California, BerkeleyRNA扩增加利福尼亚大学，伯克利.docVIP

RNA amplification protocol This method is to generate from minute amount of total RNA, sufficient amount of mRNA to perform microarray experiments You will need: Total RNA If possible use 3 μg, For a better success, clean the Trizol extracted total RNA using the RNeasy kit. To Save columns: If your plan is to pool your RNA samples. Pool first than purify - Using less than 0.5 μg of starting RNA is more challenging, you will need to perform a second round of amplification. - 1 μg is enough - More than 2 μg is theoretically better Oligo-dT-T7 primer 5 - GCA TTA GCG GCC GCG AAA TTA ATA CGA CTC ACT ATA GGG AGA TTT TTT TTT TTT TTT TTT TTT V - 3 Make a 500 ng/μl solution, use RNase free water - Must be PAGE Purification - Reference: Baugh LR, Hill A. A., Brown E. L. and Hunter CP (2001) Quantitative analysis of mRNA amplification by in vitro transcription . Nucleic Acids Research, Vol. 29, No. 5 e29. Random hexamers 5’ NNNNNN 3’ Make a 1μg/μl solution, use RNase free water Mmlv Promega 200 units/μl Good enough, cheaper than the superscript II Superscript II or III Invitrogen 200 units/μl Most used/cited in articles Is supposed to generate full length cDNAs dNTPs ISC Bioexpress, make 10 mM stocks rNTPs NEB (80 mM solutions, use as it is) E.coli DNA polymerase NEB, 10 units/μl, works with NEB buffer #2 E.coli Rnase H Takara (2150A), 60 units/μl E.coli DNA ligase NEB, 10 units/μl T4 DNA polymerase NEB, 3 units/μl T7 RNA polymerase Fermentase, 200 units/μl Pyrophosphatase Fermentas, 0.1 units/μl HEPES 1M pH 7.5 Spermidine 100mM Make 100 mM stocks, store at -20C Dithiothreitol (DTT) 1M Make 1M stocks, store at -20C Acetyled BSA 10mg/ml (100X) RNeasy kit Qiagen PCR qiaquick purification kit Qiagen RNase free Water Use the RNase free water that comes with the RNeasy kit Filter tips Speed vacuum Is in the Genelab PCR machine Incubate your reaction in 0.2 ml tubes Set the heated lid at 37C when performing the overnight invitro tran