The PDE activity in the protein fraction from our modified protocol was similar to that of homogenized ovaries not subjected to RNA isolation. In contrast, extracts from the RNA isolation kits we tested, including a kit claiming to preserve proteins in their native state, had no PDE activity or very low activity (Table 2). This may be caused by RNase-inhibiting buffer constituents inhibiting PDE activity due to their similarity to PDE substrates. There was a tendency to a slightly lower caspase-3 activity compared with no RNA purification when using the modified protocol, while the other commercial kit tested had significantly lower activity. Similar results were obtained in an LDH activity assay.
We were unable to estimate the RNA quality in the traditional way since no ribosomal RNA was present in the purified samples, precluding gel electrophoresis of the isolated RNA. Instead we used an approach utilized for microarray (13) and RNA (14-16) quality control using real-time PCR with two probe-primer-pairs located at the 5′ and 3′ ends of Gapdh and cDNA reverse transcribed with oligo-dT primers. As the reverse transcriptase always starts from the oligo-dT-primed 3′ end, increased degradation or RNA breakage will lead to a loss of signal from the 5′ end and thus an increase in the 3′:5′ ratio. We found no differences in the Gapdh ratio between the different kits (Figure 1C), although a tendency was seen for a higher mean ratio in the native protein protocol. This was primarily caused by a single outlier with a 3′:5′ end ratio of 10. As previously observed (14, 16), the average 3′:5′ ratio was above 2. This is likely due to inefficiency in the reverse transcription reaction (14, 17, 18).
A common occurrence with silica-gel spin-columns is contamination of the isolated RNA with genomic DNA (gDNA) unless preventive steps are taken (e.g., pretreatment with a DNA capture spin-column or DNase treatment). Such contaminating gDNA can cause artifactual results in RT-PCR assays (19), and in order to monitor the degree of gDNA contamination, we used two TaqMan primer/ probes (Gapdh and Rpl32) that also amplifygDNA. The degree of contamination was calculated by subtracting the Cq obtained from cDNA generated with reverse transcriptase from the Cq of the control reaction without transcriptase. It is evident from Figure 1D that there was a minimal amount of contamination in the two oligo-dT-based protocols (NP and DD) and the DNase-treated samples (QR). In contrast, a high amount of gDNA was observed in the spin-column-based protocols without DNase treatment (QA, QR, and SG) unless Trizol pre-purification was applied (T).
While the current protocol uses magnetic beads, it should be possible to use oligo-dT-coated microplates or biotin-labeled oligo-dT as long as the temperature is kept low during hybridization. Instead of eluting the mRNA, the beads can be used to create a solid-phase cDNA library or for on-bead qPCR (20, 21). Another potential modification includes the use of LNA oligo-dT to increase the hybridization rate at low salt concentrations. This would allow the use of guanidine thiocyanate in the initial wash step for more efficient denaturation of carry-over RNase contamination (22). The current amount of oligo-dT beads should be sufficient for purifying at least 4 µg of total RNA and may need to be adjusted if the amount of RNA differs significantly from this figure. This calculation is based on a binding capacity of 1 µg mRNA per 20 µL (100 µg) magnetic beads, which corresponds to at least 4 µg total RNA, assuming a 5-fold binding capacity excess is required and 5% of thetotal RNA is mRNA (20). The cost of the modified protocol is about 75% less per sample compared with the existing commercial kit for simultaneous purification of total RNA and native protein, based on the price for 500 samples.
There are some limitations to the proposed protocol. There is a tendency to a slightly lower RNA yield compared with the standard magnetic bead protocol, with a Cq difference of 0.7, although the yield is still higher than when splitting the lysate in half. It is not currently known how well the new protocol will work with tissues containing high amounts of RNase, such as pancreatic tissue. It may be necessary to shorten the hybridization time and/ or add selective RNase inhibitors to the lysis buffer, depending on compatibility with the downstream protein assay, or increased LiDS concentration in the initial washing buffer to denature carry-over RNase. In addition, compared with the spin-column based kits, the Cq values for both Gapdh and Rpl32 were lower with both oligo-dT bead protocols while Inha was not different. Consequently the normalized Inha ratio was higher for the spin-column kit. A possible explanation could be that the RNA was degraded in the spin-column kits as Gapdh is more sensitive to degradation compared with Inha (23) or that differences in poly-A tail length affect the capture rate when using oligo-dT beads (22, 24).
In conclusion, the present protocol makes it possible to carry out a simultaneous purification of mRNA and native protein from samples containing small cell numbers using a modification of the existing protocol for oligo-dT-bound paramagnetic beads. This is particularly useful when the starting amount of material is low, or the correlation between mRNA and protein activity is being investigated, for example, to cross-validate the data.
T.S.P. was involved in the conception and design of the study, data acquisition, analysis and interpretation, drafting of the manuscript, and revision of the article. C.Y.A. was involved in the design of the study and interpretation of the data, drafting of the manuscript, and revision of the article.
The excellent technical help from Christian Møller Olsen is greatly appreciated. I would like to thank my colleges Martin Stahlhut, Johan Selmer, Birte Thoke-Jensen, and Ninette Winther Hansen at LEO Pharma for constructive input and help with the phosphodiesterase assay.
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