Notes:
- Quality control for first-time users:
1) Check the total RNA for integrity and concentration (all samples). rRNAs should be clear and no degradation products visible.
2) Check presence of poly A+ RNA on gel controlling size distribution and enrichment level (some rRNA always remains).
3) Double stranded cDNA should show a size distribution between about 0.5 - 5KB with the bulk of the smear around 1.5 KB. Some tissues produce prominent transcripts that appear as bands in the cDNA.
4) Complete restriction enzyme digestion can be monitored using control DNA.
5) Ligation of anchors (adaptors) can be best monitored by PCR of the primary template (ligation mix) and should yield a strong smear between 600 bp - 50 bp.
- Apart from the total-RNA that should also be checked photospectrometrically, the assessment of the material can be done on agarose gel electrophoresis. Although these checks are a good idea in the beginning later, only an analysis of the "pre-amp" is necessary (the amplification of the primary template with primers lacking selective extensions).
- Another restriction enzyme pair (REP) commonly used with good results is EcoRI/MseI. Data from our lab and others indicates that a wide range of REPs yield similar fingerprints in terms of quality and fragment numbers.
- To obtain just one TDF per transcript a restriction enzyme with a 5-bp recognition sequence can be used. The protocol involves cutting with the 5-bp cutter and then capturing the 3’-ends of the cDNAs on magnetic beads. After elimination of the free 5’-fragments TDFs can be released from the beads with the frequent cutter. This method can also achieve a higher transcript visualisation level.
- Protocols for verification of band identity are available (see Ron van der Hulst or Bart Brugmans) and should be used where TDFs are to be isolated from gels.
- Very small quantities of RNA can be successfully used as a starting point for cDNA-AFLP. The amounts mentioned in the protocol should however be proportionally scaled down. Scaling down the procedure also allows the use of micro-titre pate (96 well) format making high throughput applications possible.
- All glassware, disposables and solutions that come into contact with RNA should be RNase free. Autoclaving usually gives enough protection and DEPC treatment or use of RNase inhibitors is unnecessary.
6. Buffers:
– 2 x STEX (autoclave) 2.0 M NaCl
20.0 mM Tris-HCl
2.0 mM EDTA
0.2 % Triton X-100 pH 8.0
– RNA extraction buffer (autoclave) 100.0 mM Tris-HCl
100.0 mM LiCl
10.0 mM EDTA
1.0 % SDS pH 8.0
– 10 x cDNAII buffer (use sterile components; filter sterilise) 200.0 mM Tris-HCl
750.0 mM KCl
100.0 mM (NH4)2SO4
50.0 mM MgCl2
10.0 mM DTT pH 7.5
– R/L buffer (x5) (10x)-“One-For-All” buffer 500 μl
BSA purified (10mg/ml) 25 μl
DTT (1M) 25 μl
Milli-Q 450 μl
7. Selective amplification with IRD 700 / 800 labelled primers
IRD 700 IRD 800
20x / 50x dilution secondary template 5.00 μl 5.00 μl
Labelled-Primer 1 (AseI (+2), 1 pmol /μl stock) 0.5 μl 0.6 μl
Primer 2 (TaqI (+2), 50 ng/μl stock) 0.3 μl 0.3 μl
10x PCR buffer (Superbuffer, SphaeroQ) 1.0 μl 1.00 μl
dNTPs (5 mM) 0.40 μl 0.40 μl
Taq-Pol (SuperTaq 5U/μl; SphaeroQ) 0.04 μl 0.04 μl
H2O 2.80 μl 2.70 μl
---------- ----------
10.00 μl 10.00 μl
Touch-down profile:
(94ºC, 30 sec; 65 – 56ºC (decrease 0.7ºC each cycle), 30 sec; 72ºC, 60sec) x 12
Continue with :
(94ºC, 30 sec; 56ºC, 30 sec; 72ºC, 60 sec) x 24
