Real-time quantitative PCR : A fast and efficient method to screen clones derived from Subtractive Suppression Hybridization

C Louvet
INSERM U437 - ITERT
30, bd Jean Monnet
CHU-HOTEL DIEU
44093 NANTES
telephone: (33)2 40 08 74 10
fax: (33)2 40 08 74 11
email: clouvet@nantes.inserm.fr
prestype: Platform
presenter: Louvet C

Louvet C, Heslan JM, Chiffoleau E, Cuturi MC.

Introduction. Subtractive Suppression Hybridization (SSH) has proven successful identification of differentially expressed genes between two types of cells or between two conditions. SSH technique uses selective exponential amplification by PCR to isolate target cDNA fragments. As a result, the subtracted library can contain rare differentially expressed transcripts enriched by ~1000-fold. However, SSH from high-complexity cDNA samples may generate numbers of background clones representing non-differentially expressed species, even after primary screening by differential hybridization. Northern blotting is generally used to confirm differential expression but this method remains semi-quantitative and often not sensitive-enough to detect low-abundant mRNAs. To overcome these problems, we introduced real-time quantitative PCR to precisely assess the expression levels of clones derived from SSH.

Materials and Methods. Acute rejection of heart allograft in an adult rat model can be prevented by donor-specific transfusion (DST) of the recipient before the engraftment. To identify novel genes involved in tolerance induction, we performed SSH on mRNAs from whole-heart grafts of untreated and DST-treated animals, followed by screening by differential hybridization with subtracted probes, sequencing, and SYBR® Green real-time quantitative PCR on cDNA pools of syngenic, allogenic-rejecting, and non-rejecting-allogenic grafts.

Results. Among the 288 randomly selected clones from SSH, 50 putative clones showed differential hybridization with subtracted probes. Based on their sequences, primers were designed for 25 candidates. Real-time quantitative PCRs were carried out and allowed to eliminate 23 false-positive clones and retain only two true clones corresponding to transcripts specifically accumulated in the non-rejecting allografts. Expression levels of these transcripts were then assessed in grafts from day 1 to 7 after transplantation and revealed over-expression for both clones from day 3 to 7 in the non-rejected allografts.

Conclusions. SSH can be performed on high-complexity cDNA samples derived from whole tissues like heart allografts when it is coupled with real-time quantitative PCR as assesment of the true levels of mRNA expression. In addition, kinetics analysis can be concurrently done on numerous samples to rapidly and precisely determine the modulation of expression for each transcript of interest.