Download RNA or DNA Extractions: Where can I get my samples extracted

RNA or DNA Extractions:
Where can I get my samples extracted?
The VCC DNA Analysis Facility offers nucleic acid extractions as a service and can be
ordered through your iLab Solutions account
at https://my.ilabsolutions.com/service_center/show/3129?tab=services. The Microarray
Facility staff can also assist investigators with the proper techniques and reagent selection
for extracting RNA or DNA. Please email the staff or call 802-656-3936.
How do I properly prepare space for use with RNA extractions? When extracting RNA,
the work space must be properly treated to eliminate the ubiquitous RNases. It is
recommended to wipe down the area with a surface treatment agent such as RNase Zap
(Life Technologies) or RNA Away (Molecular BioProducts). Whenever possible, perform all
work in a PCR workstation. All plastics, glass, and consumables used in the process should
be purchased as RNase-free or treated with RNase Zap, rinsed with Nuclease-free water,
and autoclave or baked for 2hr. All reagents purchased should be RNase-free or made fresh
with RNase free or DEP-C treated water.
Should I add an RNase Inhibitor to my RNA sample? It is always a safe precaution to
add 10 units of RNase Inhibitor such as RiboLock from Fermentas corp (or equivalent), for
every 30 ul volume of RNA. Likewise, RNase inhibitors may also be added to wash buffers
or similar reagents that might come in contact with the cells or sample. When in question,
don’t hesitate to contact the facility staff at http://vgn.uvm.edu/microarray/personnel.
What is the best method to use for extracting RNA from Cells? The Qiagen RNeasy Mini
(>105 cells) or Micro kits (<105 cells) generally work well for eukaryotic suspended and
adherent cells, but most Silica column-Guanidium lysis reagents are fine. For samples that
are suspect of being high in extracellular biomolecules such as polysaccharide, adipose, or
similar, a silica column may perform poorly and Trizol should be used.
What is the best method to use for extracting RNA from Tissue? A TriZol extraction
using a high speed homogenizer such as the FastPrep system perfoms well. The use of an
abrasive material such as diamond or garnet will assist with the homogenization. Further
clean-up with a silica column is recommended.
What is the best method to use for extracting RNA from FACS? Please consult with the
microarray staff for successful RNA recovery from cells sorted using flow cytometry.
Several key points are highlighted below:
Preparing the flow cytometry is not a small task as it must be completely free of RNases
from the sheath tank to the sorting nozzle. This decontamination procedure will take
considerable time, so be prepared. Ensure the dip tubes, septa, flow cell, all tubing lines,
and nozzles have been completely decontaminated with bleach, RNase ZAP, ethanol,
autoclaving, or other qualifying technique prior to the sort. Rinse cytometer with DEPC
water. Remember DEPC water DOES NOT inactivate RNases, and is only RNase-free water.
The sheath fluid and tank must be RNase free. Use only sterile RNase free tubes on the
cytometry that have never been open to contaminated air.
As a control, retain some cells and extract the RNA to determine the condition of RNA prior
to the sort. Also before and after trypsinization for adherent cells. Often the flow cytometry,
even after a good decontamination procedure can be the source of RNases. Whenever
possible suspend cells in RNAlater prior to the sort and after trypsinization. Again,
checking RNA integrity at this step is a good control. If RNAlater is not an option, you may
consider adding RNase inhibitor to prevent degradation during a sort. Use RNase inhibitors
that are DTT independent such as Ambion's Superase-In.
During the sort, sort cell directly into your extraction reagent, something like Trizol LS or
Qiagen's RLT. When using TriZol, Only Use TriZol LS which is slightly more concentrated
than regular regular TriZol. This formula allows lower quantities of reagent to be used
relative to the amount of the sample. (Regular TriZol can tolerate 10 part TriZol to1 part
sample volume while TriZol LS can allows 3:1. In the case of Guanidium Isothiocyanate (or
Qiagens's RLT buffer) sort at a ratio of 100ul of sort liquid to 350 ul of RLT or a multiple of
that.
Immediately after the sort, extract RNA according to the selected reagents manufacturer's
protocol and evaluate the integrity of the RNA.
What is the best method to use for extracting RNA from LCM? Both the PicoPure and
Qiagen Micro Kit perform well. With small recovery targets, it is recommended to skip the
on-column DNase treatment to increase recovery of RNA as it is known that losses occur
during this procedure. It is also recommended to elute off the column with water of 60 C
and a slightly basic pH.
What is the best methodology to use for DNase treatment? We have always found a
DNase treatment performed on a silca gel memebrane column to be the most effective for
eliminating gDNA and void of possible cations that can be left behind and possibly inhibit
enzymatic reactions with a post RNA recovery treatment. We recommend to add twice the
amount of DNase units (27.3U) and double the incubation period (30 min) with an oncolumn treatment to insure all or most gDNA has been eliminated. If an “in tube” DNase
treatment is used (such as the Turbo Kit by Ambion), be aware that downstream
quantitation maybe artificially inflated.
How can I concentrate my RNA? RNA can be concentrated with a speed vac without
compromising the integrity if done properly. First, properly clean the speed vac with
RNaseZap and Ethanol to insure no RNases are present. On each sample tube install a
breather membrane (breathe easier tube membranes) over the tubes to minimize
introducing exogenous materials. For instructions on proper speed -vac cleaning, go to the
protocols link.
What RNA kit should I use for cleaning or concentrating RNA? This is highly
dependent on the starting concentration of the RNA you wish to clean or concentrate. To
concentrate or clean total RNA, we prefer silica column technologies (such as RNeasy mini)
are acceptable however if you desire miRNA’s or have limited RNA then RNeasy Micro kit is
ideal because it can be eluted in as little as 12 ul of 60 C water.
What about extraction of bacteria or yeast? Certainly bacteria and yeast will require
additional effort. This includes growing bacteria and yeast to no greater then early log and
performing some enzymatic treatment. For yeast we recommend lytic enzyme from Sigma
and following the RNeasy protocol. However, it is required to microscopically check the %
sphearoplasting under the microscope. Not all lytic enzymes are created equal. For a high
efficiency protocol for yeast see: Microarray Outreach Lab Manual Yeast-DMSO
It is understood that Gram negative bacteria are easier to get RNA from than Gram Positive.
In both cases we recommend a Protinease K and lysozyme treatment followed by 50 C
Trizol addition and homogenized using diamond and a the FastPrep-24.
RNA Extraction in the presence of ECM or similar Major problems can occur when
isolating RNA using silica columns from unusual matrices such as those with high ECM, fat,
or alginate as they interfere and coat the silica and render it inactive. For this purpose the
first choice is Trizol followed by a silica clean-up column.