Validation of mRNA Quantification in Human Kidney by a Novel Fluorescent Nested RT-PCR Method

Karet, Fiona E.; Charnock-Jones, D S; Harrison-Woolrych, ML; O'Reilly, GE; Davenport, A. P.; Smith, Stephen K.

doi:10.1042/cs045003p_pt2

Cited by 1 publication

(2 citation statements)

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“…Their sequences are identical to human ClC-2 at nt 2177-2195 and 2347-2369 respectively in the coding sequence (10). For evaluation of the ratio of the two forms, HRC5′ was labeled with blue fluorescent dye FAM (Applied Biosystems, Inc., Foster City, CA) (16), and 25-31 cycles or 29-37 cycles of amplification were performed depending on the yield of the PCR products. In each cycle, the reactions were denatured at 94_C for 1 min, annealed at 57_C for 40 s and extended at 72_C for 40 s. PCR products were separated on a Model 373 ABI DNA Sequencer and quantified by GeneScan software (Applied Biosystems).…”

Section: Pcr Amplificationmentioning

confidence: 99%

“…Initial agarose gel electrophoresis of the PCR products using HRC5′ and HRC3′ suggested that the brain had the highest ratio of long/short form transcript compared with other tissues, especially the lung and the L2 cells. To quantify the ratio of these long and short transcripts, RT-PCR was performed with a fluorescent-labeled oligonucleotide (16). As only one pair of primers was used in the PCR reactions and two fragments were amplified, each fragment acted as the internal standard for the other.…”

Section: Adult Rat Lung Has a Decreased Long/short Form Ratio Of Clc-mentioning

confidence: 99%

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A short CIC-2 mRNA transcript is produced by exon skipping

Chu

Murray

Liu

et al. 1996

Nucleic Acids Research

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CIC-2 is a voltage- and volume-regulated chloride channel expressed in many tissues. We have shown that CIC-2 in rat lung airways is significantly down-regulated after birth [Murray,C.B. et al. (1995) Am. J. Respir. Cell Mol. Biol., 12, 597-604]. During PCR amplification from rat lung cDNA, a second transcript was identified which is 60 bp shorter than the full length sequence. The peptide translated from this 60 bp sequence contains many positively charged amino acid residues. Rat genomic DNA sequencing showed that the 60 bp sequence is an intact exon. A 71% pyrimidine content and an AAG 3'-end splice site in the intron immediately upstream from the 60 bp sequence were identified which may account for the alternative splicing of the following exon. Human genomic sequence analyses demonstrated similar intron-exon arrangement. A high CT content and an AAG 3' acceptor site were conserved in the intron corresponding to the rat upstream intron. The presence of the full length short form transcript was confirmed in rat kidney by RT-PCR, and the ratio of the long and the short form transcripts varied significantly according to the tissues examined, with the lowest long/short form ratio found in the lung among the tissues studied. Our data demonstrated that the alternatively spliced short form (CIC-2S) is transcribed in many rat tissues, the ratio of the long/short form transcripts is lower in the lung compared with the brain, and the genomic organization in this area is conserved in rat and human.

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Section: Pcr Amplificationmentioning

confidence: 99%

Section: Adult Rat Lung Has a Decreased Long/short Form Ratio Of Clc-mentioning

confidence: 99%