Speaker 1: Successful RNA purification using the Monarch Total RNA Mini Prep Kit can easily
be accomplished by following these helpful tips.
Speaker 1: Before your prep.
It is important to work in an environment free from RNases.
Always wear gloves, use RNase-free glass and plastic wear and clean your work areas.
We recommend wiping your bench top with a cleaning agent such as RNaseZAP.
Speaker 1: To maintain RNA integrity of your sample, prior to the prep, samples should
be frozen at the time of harvest, or protected by a dedicated reagent such as the Monarch
DNA and RNA Protection Reagent, which is included in our kit.
Cultured cell pellets are aliquots of blood preserved with this protection reagent, should
be mixed thoroughly by vortexing or pipetting prior to storage.
Speaker 1: Small pieces of tissue less than 20mg may be stored directly in the protection
reagent.
However, larger tissue samples should be homogenized in protection reagent prior to storage.
If samples have been preserved in the protection reagent, do not remove the liquid prior to
processing, as the protection reagent also begins the lysis of the sample.
Speaker 1: It's important to avoid thawing your sample in order to preserve it's integrity
before extraction of the RNA.
Frozen cultured cells can be thawed briefly before the addition of the RNA lysis buffer,
but most other sample types should not be thawed in the absence of the protection reagent
or the lysis buffer.
These samples include tissues, blood, bacteria, yeast and plants.
Speaker 1: Ensure that your samples are completely disrupted and homogenized in order to release
all of the RNA and maximize your yield.
Mechanical homogenization for example, with a bead homogenizer, can boost your yields
beyond those produced by proteinase K digestion alone when you're working with tissue.
Mechanical homogenization is also recommended for tough to lyse samples such as plant, bacteria,
and yeast.
Speaker 1: When using mechanical lysis and homogenization, multiple rounds of homogenization
may be recommended.
If you do use multiple rounds, place your sample on ice for approximately one minute
in between rounds to prevent your sample from over heating.
If you are working with tissues or leukocytes it can help to preheat your heating block
to 55 degrees Celsius before starting your prep, so that the heat block will be at the
correct temperature when you get to the proteinase K incubation.
Speaker 1: Please be aware that when working with mammalian whole blood, the proteinase
K incubation is carried out at room temperature.
We also recommend preparing a master mix of DNase I and DNase I reaction buffer prior
to beginning if you are performing multiple preps at once.
Speaker 1: Be sure to follow our guidance on the recommended sample input amounts in
order to ensure that the buffer volumes are appropriate and that the columns are not overloaded.
This is a common mistake that quickly can reduce yield, purity, and integrity of your
RNA.
Speaker 1: After sample lysis, perform all steps at room temperature.
This will prevent detergent from precipitating in the buffers.
Do not place your samples on ice after lysis.
Before applying your sample to the genomic DNA removal column, be sure that you label
the collection tube, as you'll be discarding the column after you centrifuge.
After putting your sample through the genomic DNA removal column, make sure that you've
saved the flow through.
This flow through contains your RNA.
Speaker 1: If you want to capture total RNA including small RNAs, add one volume of ethanol
to your flow through from the gDNA removal column, in preparation for binding it to the
RNA purification column.
If you want to exclude RNAs smaller than two hundred nucleotides, only add half the volume
of ethanol to your flow through from the gDNA removal column.
Speaker 1: It's very important to perform all wash steps in the protocol in order to
produce highly pure RNA.
Be sure to spin your column for two minutes after the last wash.
For low yield samples, including muscle or brain, or for low starting inputs, consider
eluting the RNA with 50 microliters of nuclease free water in order to get concentrated RNA.
It's important to have a concentration above 20 nanograms per microliter.
If a microvolume spectrophotometer, such as a nanodrop, will be used to measure concentration
and purity.
Speaker 1: In some cases, silica particles from the column matrix can be found in your
eluate.
To ensure that this doesn't affect your OD260-230 ratio, you can centrifuge the eluate for one
to two minutes at 1600 x G and pipet the aliquot from the time of the liquid for measurement
on a spectrophotometer.
Speaker 1: While nucleus free water is provided in this kit for the elution of RNA, adding
EDTA to a final concentration of .1 to 1 millimolar can protect RNA samples that will be stored
for an extended period of time.
Additionally, it's good practice to store the eluted RNA in aliquots to avoid excessive
freeze-thaw cycles.
Speaker 1: We hope that these tips have been helpful.
If you have any further questions, our tech support staff would be happy to help.
Contact us at info@neb.com or online at neb.com/tech support.