and u03b29 rna seq id nos 19 amp 20 cg u03b231 u03b22 u03b23 u03b25 u03b26 u03b27 u03b28 u03b29 and lh u03b2 genomic dna seq id nos 23 amp 24 the use of the cgi primer also makes it possible to rule out amplification of any lh u03b2 rna which may be present this is because the last three bases of the cgi primer correspond to the triplet acc which encodes the threonine 42 residue of the mature cg u03b2 protein however the u03b2 subunit of lh possesses a methionine residue encoded by the triplet atg in position 42 cg u03b21 u03b22 u03b23 u03b25 u03b26 u03b27 u03b28 and u03b239 rna seq id nos 19 amp 20 1 u03bcg of total rna from each sample was reverse transcribed in a volume of 20 u03bcl using the ampli gene rna pcr kit perkin elmer in accordance with the manufacturer 39 s instructions the reaction mixture for each sample contains 20 units of reverse transcriptase 50 units of rnase inhibitor 2 5 mm oligodt 1 mm of each deoxyribonucleotide triphosphate da dt dc and dg 10 mm dtt 10 mm tris hcl ph 8 3 50 mm kcl and 5 mm mgcl2 the reaction mixtures are incubated consecutively in a u201c dna thermal cycler u201d 9600 perkin elmer for 10 minutes at 20 u00b0 c hybridization of the oligodt 30 minutes at 42 u00b0 c reverse transcription and 5 minutes at 99 u00b0 c denaturation of the reverse transcriptase and of the rnase inhibitor after which they are cooled rapidly to 4 u00b0 c the equivalent of 500.