Aging Aging

Differentially Expressed Genes 113Table 7Primer Sequence of a β-actin gene that Gives Bands of Different Sizesfor cDNA and gDNATarget Primer sequence (5' → 3') Product (bp)β-Actin sense GGC GGC ACC ACC ATG TAC CCT 202 (312)β-Actin antisense AGG GGC CGG ACT CGT CAT ACT4. Notes1. Working with RNA demands some special precautions, as RNA is rapidlydegraded in the presence of the very stable enzyme RNase. The possibility ofcontamination with RNase should be minimized. Therefore always wear gloveswhen preparing or handling RNA probes. The laboratory equipment for RNAprocedures should be separated if possible or sterilized in an autoclave beforeuse. Equipment that cannot be autoclaved should be treated with 0.5 M NaOH or70% ethanol. RNase can be inactivated with DEPC (CAUTION: carcinogen; usea fume hood). DEPC should be added to every solution in a final concentration of0.1% (but not to solutions that contain Tris) and then incubated overnight in afume hood with cover open, or if possible autoclaved for 20 min.For RNA preparation, a method for isolating cytoplasmic RNA is useful, toavoid amplification of incompletely spliced RNA in the reverse transcription.Genomic DNA contamination is one of the major causes of false-positiveresults. Although most kits guarantee preparation of DNA-free RNA, it would beuseful to check samples for possible DNA contaminations. This could be done byan RT-PCR reaction with a primer to genes usually expressed that gives differentproducts with mRNA and gDNA (e.g., Table 7).In the RNA gel electrophoresis, the formamide that inhibits the RNases can bereduced by half if the additional volume is needed.2. It is useful to confirm the success of the reverse transcriptase reaction in a simplecommon PCR reaction with primers to genes usually expressed in the material ofinterest.3. The use of too much cDNA in the amplification reaction, or in the reamplification,can have a bad effect on the reaction. In the worst case, there may be no amplificationof the DNA at all.The various bands of the acrylamide gel should be cut out as small as possible,to avoid contamination with neighboring bands.4. Silver staining is a very sensitive method for oligonucleotide detection. To beable to differentiate varying bands, the probe dilution in the acrylamide gel electrophoresishas to be optimal. Our experience has shown that 5–8 µL from theamplification reaction are enough to detect clear bands.The developing reaction in silver staining can happen very quickly. To avoidthe gel becoming too dark it is useful to prepare the stop/desilver solution before-