Total RNA was isolated from cells using Trizol (Life Technologies) and quantitated by Nanodrop. 20 μg total RNA was run on 20% (w/v) acrylamide/8M urea gels with a 32P-labeled Decade marker (Ambion), and then transferred onto Hybond-N membranes (Amersham Pharmacia Biotech). After transfer, the membrane was either UV crosslinked or EDC-mediated chemical cross-linking (Sigma)(Pall and Hamilton, 2008 (link)). Pre-hybridization was performed with PerfectHyb™ Plus Hybridization Buffer (Sigma) at 37°C for 10 min. 32P-labeled probes that reverse complement to the targeted miRNAs were hybridized with membrane overnight at 37°C. After washing with 2X SSC with 0.1% SDS buffer for 3 × 15 min at 37°C, the membrane was exposed to an Imaging Screen-K (Bio-Rad) overnight. Images were then analyzed by Typhoon Trio Imaging System (GE Healthcare).
32p labeled decade marker
Manufactured by Thermo Fisher Scientific
The 32P-labeled Decade marker is a DNA ladder used for size determination in gel electrophoresis. It consists of DNA fragments labeled with the radioactive isotope 32P, allowing for visualization on autoradiography film. The marker provides bands at regular size intervals, typically in a logarithmic scale, to enable the estimation of unknown DNA fragment sizes.
Automatically generated - may contain errors
Lab products found in correlation
Perfecthyb plus hybridization buffer, by Merck Group (3 mentions)
Hybond n membrane, by Cytiva (3 mentions)
Imaging screen k, by Bio-Rad (3 mentions)
Typhoon trio imaging system, by GE Healthcare (3 mentions)
Hybond, by Cytiva (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
3 protocols using 32p labeled decade marker
1
Northern Blotting for miRNA Analysis
2
Northern Blot Analysis of miRNAs
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Total RNA was extracted by using TRIzol reagent (Ambion). Twenty microgram total RNA and a 32P-labeled Decade marker (Ambion) were loaded into 20% (w/v) acrylamide/8 M urea gels. After gel electrophoresis, RNAs were transferred onto Hybond-N membranes (Amersham Pharmacia Biotech) using a semidry transfer apparatus, followed by either UV cross-linking using 1500J for detecting over-expressed miRNAs or EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)-mediated chemical cross-linking (Sigma) at 60 °C for 1 h for detecting endogenous miRNAs. 32P-labeled probes were hybridized with membrane overnight at 37 °C after pre-hybridized with PerfectHyb™ Plus Hybridization Buffer (Sigma) at 37 °C for 10 min. After washing with 2× SSC plus 0.1% SDS buffer for 3 × 15 min at 37 °C, the membrane was exposed to an Imaging Screen-K (Bio-Rad) overnight. Images were then analyzed by Typhoon Trio Imaging System (GE Healthcare). Northern blot results were quantified by Quantity One (Bio-Rad). The sequences of probes used in this study were listed in Supplementary Table 1 .
3
Northern Blot Analysis of miRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from cells using Trizol (Life Technologies) and quantitated by Nanodrop. 20 μg total RNA was run on 20% (w/v) acrylamide/8M urea gels with a 32 P-labeled Decade marker (Ambion), and then transferred onto Hybond-N membranes (Amersham Pharmacia Biotech). After transfer, the membrane was either UV crosslinked or EDC-mediated chemical cross-linking (Sigma) was used. 32 P-labeled probes that reverse complement to the targeted miRNAs were hybridized with membrane in PerfectHyb™ Plus Hybridization Buffer (Sigma) overnight at 37°C. After washing with 2X SSC with 0.1% SDS buffer for 3 x 15 min at 37°C, the membrane was exposed to an Imaging Screen-K (Bio-Rad) overnight. Images were then analyzed by Typhoon Trio Imaging System (GE Healthcare). NB results were processed and quantitated by Image J software.
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