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The following day the synthesized riboprobe was purified:
Detection of transcripts of Tgfbr1 variants by RNase protection analysis. Total RNA extracted from CA1109 effusion tumor cells were protected by corresponding antisense riboprobes, as indicated at the top. The riboprobes were synthesized by T7 RNA polymerase using T7 promoter-containing DNA fragment of individual variant as a template. The predicted protected fragments are 263 bp for D1 (DM1), 192 bp for D2 (DM2), 151 bp for D3 (DM3), and 90 bp for D4 (DM4). Lanes 5 and 6: the mixed riboprobes (M) were incubated with (+) or without (-) RNase A/T1, respectively. Lanes 1–4: the total RNA (10 μg aliquots) was hybridized with individual purified antisense riboprobes and the protected fragments were resolved on a 6% acrylamide/8 M urea sequencing gel. * Indicates an unidentified Tgfbr1 transcript variant in CA1109 tumor cells protected by the anti-DM3 riboprobe.
A small aliquot of the synthesized and purified riboprobe (4 μl) was run in 1% agarose gel in 1x TAE to check if both synthesis and purification steps worked properly. It could happen that the band is not well defined or exactly at the expected size because of the different migration property of the RNA compared to the DNA.
Digoxigenin labelled riboprobe synthesis - Carroll lab
There are many different ways to do in situ hybridization including probing with cDNAs, cRNAs, or synthetic oligonucleotides. There are also multiple ways of labeling these probes using radioactive (3H, 32P, 33P, 35S) or non-radioactive (biotin, alkaline phosphatase, digoxigenin, fluorescent) nucleotides. Over the past 10 years we have compared most of these methods in my laboratory and have come to the conclusion that 35S riboprobes represent the most sensitive method for the detection of mRNA in tissue sections (Table I).
The method we employ for in situ hybridization is basically a modification of the original protocol for radiolabeled riboprobes as presented by Cox and colleagues (Cox et al 1984a) and has been used by our lab for many different projects (Majesky et al 1990; Nelken et al 1991; Rosenthal et al 1987; Wilcox, 1992; Wilcox, Derynck, 1988; Wilcox et al 1989a; Wilcox et al 1989b; Wilcox et al 1988). This method can be adapted for synthetic oligomers (40-50mers), cultured cells or paraffin embedded tissues.
RNA riboprobe synthesis — English - CNR
Major topics include General Techniques for Handling Nucleic Acids, DEP treatment of solutions, Phenol Extraction, Ethanol Precipitation of Nucleic Acids, PEG Precipitation of DNA, Restriction Enzyme Digestion, RNA mRNA extraction, RNA Slot Blots, Formaldehyde Denaturing Gels for RNA, Ribonuclease Protection Assay, Primer Extention, Riboprobe Synthesis , Subcloning of DNA fragments, and many more...
Please, look at the provided tutorial “Search for planarian genes based on sequence similarity” and use the available tool “Sequence similarity search” to compare the mRNA sequence found in with the sequencing output (cloned fragment). If the correct sequence was cloned in the plasmid, the construct can be used for the riboprobe synthesis.
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GeneDetect® Riboprobe synthesis templates*
This amplified and purified DNA represents the template for the riboprobe synthesis reaction. The riboprobe synthesis reaction consists of the ON incubation (about 16 hours) at 37°C of 0.8-1 ug of purified PCR product, T7 RNA polymerase (Promega), the transcription optimized buffer (Promega), Digoxigenin (DIG)-nucleotides (Roche) and RNase Inhibitor (Promega) in Milli-Q water following the dilution suggested by the manufacturer. The nucleotide that constitute the riboprobes are labeled with DIG, in order to be able to detect the riboprobe localization with Ab anti-DIG.
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