FAQ about AquaRNA
1. How should I store the AquaRNA solution?
It may be stored at 22 °C for 12 months. If AquaRNA becomes precipitated when exposed to low temperatures, you may incubate it at 37 °C for 15-20 min to resolubilize the reagent.
2. My RNA was degraded, where was the RNase coming from?
To troubleshoot RNase contamination, you may set up a DNase I digestion in 1x DNase buffer. Before adding DNase I, divide the sample into two aliquots and add DNase I to one of them. If RNA degradation is seen only in the DNase I treated sample, your DNase I may be contaminated. If RNA is degraded without adding DNase I, your RNA sample may be contaminated by RNase. A good habit to prevent RNase contamination is to ensure your gloves not touching the mouth of the tube when opening and closing the tube containing RNA solution.
3. How should I remove the genomic DNA from my DNA/RNA preparation?
You may add 0.2 U of DNase I to 10-20 μl of DNA/RNA solution in 0.5-1x DNase buffer, incubate at 22-37 °C for 20-30 min, and then run the sample in a 0.8% native agarose gel to confirm the completion of DNA digestion. To inactivate the DNase I, you may use Ambion’s DNase removal reagent or inactivate the DNase I at 65 °C for 15 min. Additionally, large RNA, such as mRNA, may be differentially precipitated in 25% isopropanol from the AquaRNA lysate.
4. Why did my DNA/RNA solution show a strong absorption below A260?
It is likely due to trace amount of guanidine salt contamination. If it interferes with your downstream applications, you may further purify the extracted DNA/RNA with a silica spin column (e.g., a plasmid miniprep column). Simply add an equal volume of 4 M GuHCl and 1M NaOAc (pH unadjusted, ~7.0) to your DNA/RNA solution (may contain the insoluble pellet) and load it into the spin column, centrifuge to allow DNA/RNA binding to the silica membrane, wash the column with 0.6 ml 75% EtOH, and elute the DNA/RNA in 50 μl of deionized water or TE buffer.
5. Can I do RT-PCR without removing the contaminating genomic DNA?
Complete DNA removal may be difficult to achieve and unnecessary if you use intron-spanning primers for the PCR amplification. You may also design and use a 5’ tailed RT primer to make the cDNA and then use a pair of PCR primers with one of them complementary to the unique tailed region of the RT primer to amplify the cDNA [Hurteau and Spivack. mRNA-specific reverse transcription-polymerase chain reaction from human tissue extracts. Anal Biochem. 2002 Aug 15;307(2):304-15; and Chen, et al. Real-time quantification of microRNAs by stem–loop RT–PCR. Nucleic Acids Research 2005 33(20):e179], especially when intron-spanning is unavailable. In any case, you should always include a no-RT control in your amplification to confirm that your primers do not amplify the contaminating genomic DNA.