My student optimizes the RNA dot-blot method using a biotinylated cDNA probe. The aim is to detect a positive hybridization signal of a probe complementary to transcripts of a housekeeping gene for Human actin beta in the human RNA samples. So far, we have not been able to detect consistent hybridization signals. Either there were non-specific, very weak or no hybridization signals at all. Above it, we have recently observed a reverse white signal in dot-blot spots with our human RNA after colourimetric detection. The only such phenomenon I found when googling for the solution appears after chemiluminescent signal detection. The alleged ghost bands or spots appear when a sample or antibody excess consumes chemiluminescent substrates too quickly. However, in the case of colourimetric detection, the chromogenic substrate accumulates instead of being consumed, even in the excess of both sample and antibody. In search of a solution, we performed a dot-blot with a matrix of sample and antibody amount combinations, which resulted in either reverse or no signal et all. Does anybody have any idea where the problem could be?
Please, see the attached photo of the dot-blot results. There are 3 membrane strips, the numbers 1, 3, and 5 at each strip indicate the antibody dilution 1:1000, 1:3000, and 1:5000 respectively. The pink strip on the top of the photo shows the identity (and in the case of RNA samples also the total amount) of loaded samples in the indicated order. There are total RNA samples at the concentrations 5 ug, 500 ng, and 50 ng, followed by positive control spots of biotinylated lectin (positive control of colourimetric development of a signal produced by reporter molecule HRP in conjugate with streptavidin) and the rightmost spot is the labelled probe.
I will be very grateful for any suggestions!
White residue does usually mean too much signal and the chromophore has been used up but I wonder in your case if the saple has not bound to the membrane but has affected the loading spot so that it is blocked more than the rest of the filter producing no signal.
Samples are usually bound to the membrane in high salt so that everything binds and then the filter is washed at lower salt to get a strong bound signal but also a low background. I would check the type of membrane to ensure good sample binding ( heating the membrane or incubating in NaOH for some nylon membranes) Possibly use a higher salt wash to ensure getting a strong signal but possibly with high backround and if that works then try more stringent washes but getting a positive signal will make troubleshooting easier because negative results can come from many sources...wash temperature.wash or binding salt concentrations or faulty reagents
Paul Rutland Thank you so much for the useful tips! I also asked some of my colleagues who suggested it could be a problem with polysaccharide or protein contamination in our RNA samples which eventually turned out to be the problem, at least partially. However, although the reverse signals disappeared the hybridization signals were still insufficient so I was thinking that maybe the stronger salt washes as you suggested could help.
Could you also clarify the role of NaOH in nylon membranes as you suggested? Is it there only to increase the sample binding capacity of the positively charged nylon membrane? Based on your comment about NaOH I was googling a little and it seems that NaOH may contribute to better binding capacity of nylon membranes. Is this what you were implying in your comment? Thank you for your suggestions!
Well done Pavlína Věchtová that sounds like good progress.
NaOH denatures the hydrogen bonds between the complementary bases of dna or the secondary structure regions in rna and then the single stranded nucleic acid binds covalently (so cannot wash off) so what I was saying was that you would get both more binding and stronger binding of your template dna. Heat and UV radietion can also be used and one reason for using a choice of binding methods is that it may be possible that one method may block a binding site for your probe.
You can bind the probe at high salt concentration (6xssc) and then wash a filter in 2xssc just to prove that you have binding albeit against a high background and then was at a higher stringency (0.5) x SSC) to get a more specific signal once you know that you have binding both to the membrane and to the probe
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