AQUARNA

DNA. RNA. Protein. Extract All Bioanalytes Concurrently.

AquaRNA™ is a multifunctional aqueous reagent for DNA/RNA and protein extraction. This single solution will lyse the cells, inactivate degradative enzymes, and extract DNA/RNA and proteins. DNA/RNA are recovered from the cell lysate by isopropanol precipitation, while proteins remain soluble in the AquaRNA-isopropanol solution and can be recovered by acetone precipitation or dialysis. AquaRNA enables concurrent isolation of DNA/RNA and proteins from the same specimen without using different DNA, RNA, and protein extraction kits.

AquaRNA Brief Protocol

(Download AquaRNA User Manual for protocols to extract DNA/RNA/proteins from cultured mammalian and microbial cells, animal and plant tissues, and more)

1. Harvest the Cells

(A) For eukaryotic cells:

Centrifuge at 3,000 xg for 5 min to pellet 0.5-2 million cultured cells in a 1.5-ml microfuge tube, and aspirate to remove the medium.

(B) For microbial cells:

Centrifuge 0.25 ml log-phase culture at 12,000 xg for 1 min to pellet the cells and aspirate to remove the medium. Suspend the cells in 100 ul of 1 mg/ml lysozyme (use lyticase for yeast cells) in TE buffer (pH 8-9) and incubate on ice for >15 minutes to breakdown the cell walls. Centrifuge to pellet the cells and aspirate to remove the supernatant.

2. Extract the DNA/RNA

Add 100 ul AquaRNA solution to the cell pellet. Vortex vigorously for 60 sec to lyse the cells. Centrifuge at 12,000 xg for 5 min to pellet the debris.

3. Pellet the DNA/RNA

Transfer the clear lysate (80 ul) to a new 0.5-ml microfuge tube.

(A) To pellet total DNA/RNA:

Add 0.9 vol (72 ul) of isopropanol to the lysate. Vortex and centrifuge at 12,000 xg for 5 min to pellet the DNA/RNA. Transfer the protein-containing supernatant (100 μl) to a new tube for protein recovery.

(B) To pellet RNA and DNA differentially:

Add 0.25 vol (20 μl) of isopropanol to the lysate. Vortex and centrifuge at 12,000 xg for 5 min to pellet large RNA. Transfer the supernatant to a new tube. Add an additional 50 μl of isopropanol, vortex and centrifuge for 5 min to pellet the DNA and small RNA. Transfer the protein-containing supernatant (100 μl) to a new tube for protein recovery.

4. Rinse the DNA/RNA

Gently fill up the tube and its lid with 70% ethanol from a squirt bottle and then decant to discard the ethanol solution. Repeat the 70% ethanol rinse once. Flip the tube forcefully a few times and tap it on a paper towel to remove residual ethanol. Let the DNA/RNA pellet air-dry for 5-10 min. Add 100 ul of nuclease-free water to the pellet, vortex and/or pipette to solubilize the DNA/RNA pellet. Incubate at 22 °C for 5 min, centrifuge at 12,000 xg for 5 min to pellet any insoluble. Transfer the DNA/RNA solution to a new tube and store it at -20 °C.

5. Recover the proteins

Add 4 vol (400 μl) of acetone to the isopropanol supernatant (100 μl) obtained after DNA/RNA precipitation. Vortex and centrifuge at 12,000 xg for 5 min to pellet the proteins. Decant to discard the supernatant. Immediately add 100 µl ProMelt (#1150, order separately) to the wet protein pellet. Pipet and vortex to solubilize the proteins. Centrifuge again to pellet any insoluble and save the protein solution for SDS-PAGE.

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.