1. Why should I buy a Total RNA Multiple ChoiceTM Northern Blot? The total RNA blots are used for quick screening of the tissue distribution of your gene, for examining the tissue distribution of a highly expressed gene, or for determining the tissue distribution of an Expressed Sequence Tag in order to know from which library to clone the gene. 2. Why should I buy a Poly A+ RNA Multiple ChoiceTM Northern Blot? The poly A+ Northern blots are used to obtain a "picture perfect" Northern for publication purposes. These blots are also used to examine the tissue distribution of a low abundance gene and to study multiple forms of mRNA due to alternative splicing. 3. What should I do differently to remove a high background signal? There are several explanations for a high background: incomplete washing of the blot, too large a probe, the probe contains free nucleotides or too much probe was used for hybridization. Complete washing of the blot can accomplished by increasing the stringency of the washes. This is achieved by decreasing the salt concentration to 0.25 X SSC, by raising the temperature or by increasing the number of the washes. The ideal probe size is a DNA fragment of 200 to 800 nucleotides and free nucleotides should be removed from the probe with Sephadex G-50. The optimum amount of probe is <2 x 106 cpm/ml for DNA, <5 x 106 cpm/ml for oligonucleotides. 4. What should I do differently to get a better signal? It is possible that your probe has a low specific activity and you should make a new probe and try the hybridization again. The specific activity should be >5 x 108 cpm/ug. It is also possible that the probe was not denatured prior to hybridization. Be sure to boil the probe before adding it to the blot. If your probe is from a different species than the RNA on the blot, reduce the stringency of the final wash. With each probing of the blot, the ability to detect messages in low abundance will decrease. For highest signals, you should obtain a new blot.