About EZ RNA Methylation™ Kit
The EZ RNA Methylation™ Kit features rapid and reliable bisulfite treatment and conversion of cytosines in RNA for methylation analysis. The kit streamlines the three-step process for complete conversion of cytosine in into uracil. RNA denaturation and bisulfite conversion processes are combined into a single step. No buffer preparation is necessary. The RNA Conversion Reagent is provided ready-to-use: simply add the reagent to an RNA sample and incubate as indicated.Also, innovative in-column desulphonation technology eliminates messy precipitation steps, ensuring researchers obtain consistent results. The product has been designed to minimize template degradation, loss of RNA during treatment and clean-up, and to provide complete conversion of cytosine for accurate methylation analysis. Recovered RNA is ideal for RT-PCR, sequencing, library preparation and Next-Gen sequencing.
|RNA Input||Samples containing 32 ng - 3 μg of DNA-free RNA. For optimal results, the amount of input RNA should be between 0.5 - 1 μg.|
|Conversion Efficiency||> 99% of non-methylated C residues are converted to U with > 99% protection of 5-methylcytosine.|
|RNA Recovery||> 80%|
|Sequencing results following bisulfite treatment. RNA with methylated C (5-mC) at nucleotide position #7 was processed using the EZ RNA Methylation™ Kit. The recovered RNA was amplified by RT-PCR and then cloned and sequenced. The methylated cytosine at position #7 remained intact while the non-methylated cytosines at positions #3, 4, and 13 were completely converted into uracil (post-bisulfite treatment) and detected as thymine following RT-PCR and sequencing.|
|Step 1||Add 130 µl of RNA Conversion Reagent to 20 µl of RNA sample in a PCR tube. Mix the sample by flicking the tube or pipetting up and down, then centrifuge briefly to ensure there are no droplets in the cap or sides of the tube.
Note: If the sample volume is less than 20 µl, compensate with DNase/RNase-Free Water.
|Step 2||Place the PCR tube(s) in a thermal cycler and perform the following steps:
|Step 3||Place a Zymo-Spin™ IC Column into a Collection Tube and add 250 µl of RNA Binding Buffer to the column.|
|Step 4||Load the sample (from Step 2) into the Zymo-Spin™ IC Column containing the RNA Binding Buffer and mix by pipetting up and down.|
|Step 5||Add 400 µl of 95-100% ethanol to the sample-RNA Binding Buffer mixture in the column. Close the cap and immediately mix by inverting the column several times.|
|Step 6||Centrifuge at full speed (≥10,000 x g) for 30 seconds. Discard the flow-through.|
|Step 7||Add 200 µl RNA Wash Buffer to the column and centrifuge at full speed for 30 seconds.|
|Step 8||Add 200 µl of RNA Desulphonation Buffer to the column and let stand at room temperature (20°C – 30°C) for 30 minutes. After the incubation, centrifuge at full speed for 30 seconds. Discard the flow-through.|
|Step 9||Add 400 µl RNA Wash Buffer to the column and centrifuge at full speed for 30 seconds. Repeat the wash step with an additional 400 µl RNA Wash Buffer.|
|Step 10||Centrifuge the Zymo-Spin™ IC Column in an emptied Collection Tube at full speed for 2 minutes. Remove the Zymo-Spin™IC Column carefully from the Collection Tube and transfer it into an RNase-free Tube.|
|Step 11||Add ≥10 µl of DNase/RNase-Free Water directly to the column matrix and let stand for 1 minute at room temperature. Centrifuge at full speed for 30 seconds. The eluted RNA can be used immediately or stored at -20°C for up to 3 months. For long-term storage, keep at or below -70°C.|
Researchers from Europe showed that reduced levels of conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms, and flies. They used the EZ RNA Methylation™ Kit to show that the methylation at C2278 of 25S ribosomal RNA in yeast, worms, and flies is highly conserved, suggesting that it is important for core ribosomal function. They also observed better reproducibility and higher conversion rate using Zymo Research’s EZ RNA Methylation Kit.
Researchers used the EZ RNA Methylation Kit from Zymo Research to investigate the methylation status of several important non-coding RNAs, including XIST and HOTAIR. They found that both XIST and HOTAIR contained the 5-methylcytosine modification, suggesting that RNA methylation may play an important role in regulating the activity of these RNA molecules.