Abstract
During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosomes can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be size-fractionated by sucrose density gradient centrifugation. The more specific mRNA in polysomes implies more active translational status of the mRNA.
Materials and Reagents
Equipment
Procedure
Figure 1. MDM2 regulates MYCN translation. A. Representative polysomal profiles from control vector- and MDM2/166A-transfected SK-N-SH cells. The 254-nm traces obtained during collection of fractions are shown. B. Relative distributions of MYCN and GAPDH mRNA in SK-N-SH cells transfected either with vehicle or MDM2/166A.
Recipes
Acknowledgments
This work was supported by the National Institutes of Health (R01 CA123490 and R01CA143107 to MZ) and CURE (MZ and LG).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Hi, Sara, we used the kit "iScript? Reverse Transcription Supermix for RT-qPCR" from Bio-rad for reverse transcription, please see the link for more detailed information:https://www.bio-rad.com/en-us/sku/170-8840-iscript-reverse-transcription-supermix-for-rt-qpcrGood luck
Hi,Agata, it's a good question. but you don't worry about the downstream applications of RT,RT-PCR,qPCR,because RNA will be precipitated during RNA extraction(step:"21"),and all solutions will be washed away totally(step 24-27). RNA will be very pure and without any heparin any longer.We need heparin during cell lysis, because the DNA binding sites on RNA polymerase can be occupied by heparin, preventing the polymerase from binding to promoter DNA.