Plant total RNA used for RNA gel blotting was extracted by the hot-phenol method as previously described (Fernández et al., 1997 (link)). For the detection of viral RNAs, three 1-kb fragments at the 3′-terminus of each cDNA clone (35S-R1, 35S-R2, and 35S-R3) were amplified, which were then labeled with [a-32P] dCTP using the Rediprime II system (GE Healthcare, RPN1633) and were mixed as probes. For the detection of siRNAs, 30 mg of total RNA was separated on 17% polyacrylamide-8 M urea gels. The probes were labeled with [r-32P]ATP using T4 PNK (NEB, M0201V). VsiRNAs were detected using mixtures of labeled DNA oligonucleotides specific to RNA3. Signal intensity was quantified using ImageQuant TL software (GE Healthcare).
Rediprime 2 system
Manufactured by GE Healthcare
The Rediprime II system is a laboratory equipment designed for the random prime labeling of DNA fragments. It automates the process of generating radioactively or fluorescently labeled DNA probes, which are essential tools for various molecular biology techniques such as Southern blotting, Northern blotting, and in situ hybridization.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant tl software, by GE Healthcare (1 mentions)
T4 pnk, by New England Biolabs (1 mentions)
Storage phosphor screen, by GE Healthcare (1 mentions)
Aceq qpcr sybr green master mix, by Vazyme (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Hiscript 2 q select rt supermix, by Vazyme (1 mentions)
Amersham typhoon, by GE Healthcare (1 mentions)
2 protocols using rediprime 2 system
1
Total RNA Extraction and Viral RNA Detection
2
Comprehensive RNA Analysis Protocol
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Total RNA was isolated using the RNAprep Pure Plant Kit (TIANGEN). The cDNA was synthesized using HiScript II Q Select RT SuperMix (Vazyme). Quantitative PCR was performed with AceQ qPCR SYBR Green Master Mix (Vazyme). The relative gene expression level was normalized to AtACT2 or VdElf1. RNA blotting assay was performed as reported previously (62 (link)). For mRNA blotting, 10 µg of total RNA was separated by electrophoresis on 1.2% agarose gels with formaldehyde and transferred to nylon N+ membranes through capillary transfer. GFP DNA probe was labeled with [α-32P] dCTP using the Rediprime II system (GE Healthcare). For sRNA blotting, 60 µg of total RNA was separated by electrophoresis on 17% PAGE gels and electrically transferred to nylon N+ membranes. [γ-32P] ATP was used to label gene-specific oligo sequences by polynucleotide kinase (NEB) for hybridization (8 (link)). After washing and drying, the membrane was exposed to a storage phosphor screen (GE Healthcare). Images were scanned using an Amersham Typhoon (GE Healthcare).
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