RNA ( and optional DNA ) extraction from environmental samples ( filters ) .
Take frozen tubes out of the - 80C freezer , keep on ice .
While filtere are thawing , add RNase / DNase - free 0.5mm Silica beads to each sample tubea .
If RLT + buffer ( with beta mercaptoethanol ) was not added previously , add it here .
Vortex for 5 minutes to ensure beads disrupt filtera .
Ensure filter remains in RLT + buffer the whole timeb .
Options to increase yield :
i . Add pre - heated RLT + buffer ( heat for 2 - 3 minutes at 65C )
ii . Place tube on Tissue lyser for bead beating
iii . Vortex for additional minutes Once thoroughly lysed ( note foam / bubbles may have appeared ) , transfer liquid lysate to new tube ( avoid transferring filter ) .
Using sterile forceps , transfer the filter carefully into a 5mL sterile syringe .
Squeeze out excess lysate from filter through the syringe into the new tube of lysate .
New tube for each sample should only contain lysate .
a . Optional step is to run this lysate through a Qiashredder for additional lysis .
* * Start with Qiagen extraction steps here ( following Qiagen DNA / RNA All prep kit ) :
Transfer the lysate to the AllPrep DNA spin column .
Centrifuge for 30 seconds at > 10,000 rpm .
Transfer flow - through ( filtrate ) into a new tube for RNA purification .
This DNA column can now be stored in the fridge until DNA extraction ( 4C ) .
a . If multiple spins of the lysate are required , continue this until all lysate has been passed through the DNA column and all flow - through has been obtained in a new tube .
Add 1 volume of 70 % EtOH to the RNA flow - through product , mix by pipetting .
Do not centrifuge .
Add 1 volume of 70 % EtOH to the RNA flow - through product , mix by pipetting .
Do not centrifuge .
Transfer up to 700µl of sample ( including any precipitate ) to an RNeasy spin column .
Centrifuge for 30 seconds at > 10,000 rpm .
Discard the flow - through .
Add 350µl of Buffer RW1 to RNeasy spin column .
Centrifuge for 15 seconds at > 10,000 rpm .
Discard the flow - through .
Make up DNase I and Buffer RDD stock mix for DNase digestion .
For each sample add 10µl of DNaseI stock to 70µl of Buffer RDD , mix solution and centrifuge briefly .
Add DNaseI mix ( 80µl ) directly to RNase spin column .
Incubate at room temperature for 15 minutes .
Again , add 350µl of Buffer RW1 to RNeasy spin column .
Centrifuge for 15 seconds at > 10,000 rpm .
Discard the flow - through .
Add 500µl of Buffer RPE to RNeasy spin column .
Centrifuge for 15 seconds at > 10,000 rpm .
Discard the flow - through .
Again , add 500µl of Buffer RPE to RNeasy spin column .
Centrifuge for 2 minutes at > 10,000 rpm .
Discard the flow - through .
Option to place RNeasy spin column into a new 2mL collection tube and centrifuge at full speed for 1 minute – this will eliminate any possible carry over of Buffer RPE .
Place RNeasy spin column into a new 1.5ml collection tube , add 30 - 50µl RNase - free water .
Centrifuge for 1 minute at > 10,000 rpm to elute the RNA .
Options to increase yield :
i . Pre - heat RNase - free water ahead of addition to RNeasy column
ii . Let RNase free water sit on RNeasy column for 1 - 2 minutes before centrifugation
iii . Transfer eluted RNA back into the RNeasy column and re - centrifuge to increase concentration .
Genomic DNA purification Grab the DNA spin columns out of the fridge .
Add 500µl Buffer AW1 to AllPrep DNA spin column .
Centrifuge for 15 seconds at > 10,000 rpm .
Discard the flow - through .
Again add 500µl of Buffer AW2 to the DNA column .
Centrifuge for 2 minutes at full speed .
Discard the flow - through .
Place DNA column into a new 1.5ml collection tube , add 50 - 100µl of Buffer EB to the column .
Let sit at room temperature on the column for 1 minute .
Centrifuge for 1 minute at > 10,000 rpm .
Options to increase yield :
i . Pre - heat EB buffer water ahead of addition to DNA column
ii . Transfer eluted DNA back into the RNeasy column and re - centrifuge to increase concentration .
QC both DNA and RNA product using both Qubit fluorometer ( concentration ) and Agilent Bioanalyzer ( quality ) .
a . For the Qubit load 2µl of product to the 198µl of buffer / dye solution for each sample
