Author/Editor     Benčina, Mojca; Legiša, Matic
Title     Non-radioactive multiple reverse transcription-PCR method used for low abundance mRNA quantification
Type     članek
Source     Biotechnol Tech
Vol. and No.     Letnik 13
Publication year     1999
Volume     str. 865-9
Language     eng
Abstract     A non-radioactive multiple reverse transcription-PCR method for the relative quantitative analysis of low abundance mRNA is described. Chemiluminescent detection of cDNA products amplified by PCR and incorporating biotin-labelled dCTP was used for analysis of specific gene expression. After determining the optimal concentrations of RNA and cDNA, the tube-to-tube variations were minimised by transcription and amplification of the endogenous standard mRNA coding for protease C from Aspergillus niger, together with the transcript of interest. All the obtained data were relative values comparing to the internal standard pepC. By the method the difference in mRNA level between the two transformants with different pkaC gene copy number coding for cAMP-dependent protein kinase (PKA) catalytic subunit from A. niger was detected which was in accordance with the diferences in specific PKA enzymatic activity between both strains.
Descriptors     ASPERGILLUS NIGER
RNA, MESSENGER
CYCLIC AMP-DEPENDENT PROTEIN KINASES
GENE EXPRESSION
DNA, COMPLEMENTARY
POLYMERASE CHAIN REACTION