Cdna-AFLP Protocol

cDNA-AFLP based genetical genomic in cotton fibers, Theoretical and Applied Genetics, Michel Claverie, Marlène Souquet, Janine Jean, Nelly Forestier-Chiron, Vincent Lepitre, Martial Pré, John Jacobs, Danny Llewellyn, Jean-Marc Lacape. Corresponding author J.-M. Lacape, UMR-AGAP, CIRAD, Avenue Agropolis, 34398, Montpellier Cedex 5, France; , Tel.: +33 4 67 61 44 71

cDNA-AFLP protocol

The cDNA-AFLP protocol used was a modification of the original procedure of Bachem et al.(1996). This technique as further detailed in Vuylstekeet al.(2007) is characterized by (i) the generation of a single cDNA fragment for each messenger (‘one-gene-one-tag’) originally present in the sample (Breyne et al., 2003), (ii) the combination of a five-cutter and a four-cutter restriction enzymes, (iii) the production of cDNA-AFLP bands that are derived from the 3’-end of the gene. Three enzyme combinations were originally tested: ApoI /MseI, TaqI / MseI and BstYI /MseI. The BstYI/MseI combination was chosen because it produced an optimal density of bands after polyacrylamide gel electrophoresis (using BstYI-C+1 and MseI +2 primers). The protocol of Vuylsteke et al.(2007) was modified as follows: reduction of the number of PCR cycles during pre-amplification and selective amplification runs, and higher amounts of DNA template (ligation or PCR product) used in the PCR as detailed below.

mRNAs were isolated from 4 µg total RNA using 10 µl oligo(dT)25 magnetic Dynabeads (Dynal, Oslo, Norway). After washing steps, first strand cDNA was synthesized using the bead anchored oligo(dT) as primer and 200 U of RevertAid M-MuLV reverse transcriptase (Fermentas, Canada) according to the manufacturer’s instructions. Second strand cDNA was then synthesized in Pol1 buffer using 7.5 U of Pol1 (Fermentas), 0.01 U of RNAse H (Fermentas) and 2.5 mM dNTP at 15°C for 10 h. After washing in restriction buffer, double stranded cDNAs (anchored to the beads by the poly-A end) were then digested with 20 U of BstYI for 1 hour at 60 °C, washed three times in restriction buffer, and digested with the second enzyme MseI (20 U) for 1 hour at 37°C. After removal of the beads, the released fragments were then ligated to adapters (BstYI-Forw: 5'-CTC GTA GAC TGC GTA GT-3'; BstYI_Rev: 5'-GAT CAC TAC GCA GTC TAC-3'; MseI-Forw: 5'-GAC GAT GAG TCC TGA G-3'; MseI-Rev: 5'-TAC ATC AGG ACT CAT-3') in 40 µl final volume and in presence of 5 U of T4 ligase (Fermentas) and 10 U of BstYI. Pre-amplification was performed in 50 µl final volumes using 5 µl of the ligation product, 1 U of Taq polymerase (Invitrogen), 0.25 mM dNTPs, 4 mM MgCl2, and 0.3 µM of the MseI primer (Mse0: 5'-GAT GAG TCC TGA GTA A-3') and the BstYI primer carrying a selective base C at the 3' end (BstC0: 5'-GAC TGC GTA GTG ATC C-3'). Cycle conditions were: 94°C for 5 min and then 94°C for 30 sec, 56°C for 1 min, 72°C for 1 min (20 cycles), and 72°C for 5 min.

After pre-amplification, the mixture was diluted 20 fold and 5 μl was used for selective amplification with the 64 selective primer combinations. The BstYI primer contained one selective nucleotide and the MseI primer contained two selective nucleotides. For the 10 dpa experiment, only 62 selective primer combinations were tested. Selective amplifications were performed in 20 µl final volumes with 1 U of Taq polymerase, 0.25 mM dNTPs, 4 mM MgCl2, 0.3 µM of MseI +2 primer, and 0.05 µM of the γ33P-radiolabeled BstYI-C+1 primer. Touch-down PCR conditions for selective amplifications were: 94°C for 2 min, followed by 94°C for 30 sec, 65°C for 30 sec, 72°C for 1 min (17 cycles, with the annealing temperature reduced 0.5°C per cycle); then 94°C for 30 sec, 56°C for 30 sec, 72°C for 1 min (12 cycles).

In order to simultaneously electrophorese all of the studied RILs and parents on the same gel (94 slots) and to allow for controls and the marker ladder, only a subset of 88 RILs could be analyzed and these were chosen on the basis of their genomic content (RILs that were heavily biased in their allelic content were discarded) as were those for which the quantity or quality of the RNA were low. Two µl of amplified product were loaded on a single 5% denaturing polyacrylamide gel run for 2 h at 70 W in 0.5x TBE. Gels were dried onto 3 MM Whatman paper on a Gel Dryer Model 583 (Bio-Rad) and exposed to LifeRay films (Ferrania, Italy) for 7-10 days, which allowed visualization of the finest bands and minimized the number of saturated bands. The migration of the cDNA-AFLP bands, later referred to as transcript-derived fragments (TDFs) were compared to a 30-330bp size reference marker (Invitrogen). The sizes of the fragments larger than 330 bp were therefore only approximate.

Bachem C.W., van de Hoevan R.S., de Bruijn S.M., Vreugdenhil D., Zabeau M., Visser R.G. (1996) Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J. 9:745-753.

Breyne P., Dreesen R., Cannoot B., Rombaut D., Vandepoele K., Rombauts S., Vanderhaeghen R., Inzé Z., Zabeau M. (2003) Quantitative cDNA-AFLP analysis for genome-wide expression studies. Mol. Genet. Genomics 269:173-179.

Vuylsteke M., Peleman J., van Eijk M.J.T. (2007) AFLP-based transcript profiling (cDNA-AFLP) for genome-wide expression analysis. Nature Protocols 2:1399-1413.