Protocol to prepare RNA for cDNA synthesis followed by realtime quantitative PCR
*ALWAYS use EtOH (or Chloroform) that has Pro Analysis quality, so NOT the 100%
“histology” absolute EtOH!!!
*ALWAYS were gloves (and do not touch face/hairs/whatever with these gloves), rather
change more frequently.
*Remember that RNAses are inactivated at very high temperatures >200° C, thus if
autoclaving tubes and water eases your mind, please do so, but you can also work with
your own (fresh) bag of tubes/pipet-tips. As somebody once said: “the worst microbes are
the imaginary microbes.”)
*If working with DEPC-water remember that it is toxic, and needs to be inhibited by
autoclaving thoroughly, otherwise your enzymatic reactions are hampered!
• Isolate RNA with RNeasy-kit (Qiagen) or GTC or Trizol. For the latter, precipitate and
wash carefully with 75% EtOH (rather 2x), air-dry pellet.
Column-isolations (Rneasy) have their cut-off at ~200 nts, thus you will loose small
transcripts, and the 5S-ribosomal band!
RNA isolation
Best to carry out at RT, only centrifugation at 4° C.
•Grind fresh or ice-cold (-80° C) tissue in 600 µl Trizol (pink).
•Spin down shortly (1 min at 14,000 g) to pellet any remaining cell debris, grind again.
•Add 20% (120 µl) Chloroform, and shake/vortex, then
•spin 10 min at 14,000 g (4° C), stop centrifuge in ‘slow mode’, so you don’t diturb the
interphase. The upper phase contains the RNA, the interphase proteins and genomic
DNA.
•Transfer the upper phase to a new tube, be careful to take of ~ 250-300 µl max, and not
to take the interphase. In case you don’t do an additional DNAse treatment, it is advisable
to perform a Chloroform extraction at this stage (see chloroform extraction at
DNAsetreatment), and to continue with the steps below.
•Add 0.6 volumes of isopropanol, shake to mix and leave at RT for 20-30 min (do not put
in the freezer!)
•Spin 15 min at 14,000 g (4° C).
•Take off supernatant (trash), don’t disturb the pellet.
•Wash with ~100 µl 70% EtOH, spin shortly to get the pellet at the bottom.
1
Protocol to prepare RNA for cDNA synthesis followed by realtime quantitative PCR
•Remove all supernatant and dry the pellet at RT (~10 min).
•Resuspend pellet in the desired amount of water (or 50 µl, when continuing with the
DNAse treatment).
DNase-I treatment
• Resuspend pellet in ~50 µl H2O (when yield is >10 µg) (heat to dissolve more easily, 5 min
50-70 °C)
• Keep 2 µl behind for Bioanalyzer (RNA chip) or to put on a 1% TBE-agarose gel (put
Ethidium bromide also in the buffer, do not run too long!)
• Perform DNase treatment (in a total reaction volume of 100 µl) on remaining 48 µl, and
add:
44.7 µl H2O
3.3 µl 3M NaAc pH 5.5
1.0 µl 0.5M MgSO4
1.0 µl RNAguard/RNAsin
2.0 µl DNAse-I (Roche # 776 785)
• Incubate for 15-20 min at 37 °C.
• Add 80 µl phenol (Tris-buffered pH8.0; FLUKA) (do NOT add Chloroform at this step)
• Vortex, spin 14,000 rpm 10 min 4 °C.
• Transfer upper phase to a fresh tube (when you have little amount of RNA, add 80 µl to
the tube with phenol, vortex and spin again, and add the upper phase to the tube
containing the first upper-phase).
• Add to this tube 50 µl Chloroform, vortex, spin ~14,000 rpm 2 min (temperature is not
important)
• Set pipet at 100 µl and take off the chloroform-phase from the bottom of the tube and
leave a few µls of Chloroform.
This enables you to remove more of the upper phase (= more RNA) without the
effort of pipetting in the Chloroform-bubble that arises when you take off too
much).
• Spin again for 2 min
• Transfer upper phase to a fresh tube (when you have little amount of RNA, add 50 µl to
the tube with Chloroform, vortex and spin again, and add the upper phase to the tube
containing the first upper-phase).
2
Protocol to prepare RNA for cDNA synthesis followed by realtime quantitative PCR
• Precipitate with 0.1 volume 3M NaAc pH 5.5, and 2.5-3 volumes of EtOH (and
glycogen or linear acrylamide when desired) in -20 °C or on ice (15-30 min).
• Spin 14,000 rpm for 20-30 min (use 30 min when precipitated on ice)
• Wash pellet carefully with 75% EtOH, air-dry.
Bioanalyzer/agarose-gel analysis
• Resuspend DNase-treated pellet by adding 48 µl H2O (alternatively to a concentration
of ~200 ng/µl), heat sample 5 min at ~70 °C (easier to dissolve and RNA is stretched
immediately which is better for analysis on gel or by Bioanalyzer), and vortex or pipet
well
• Load 1 µl DNase-treated sample (and the non-DNase treated samples) on Bioanalyzer
chip [12 samples (1 µl each) can be loaded on one chip (45€/chip)]
Figure 1: Intact (lane 1, and 3) and degraded (lane 2) RNA on an agarose gel (0.1x TAE)
lane:
1
2
3
3
Protocol to prepare RNA for cDNA synthesis followed by realtime quantitative PCR
Figure 2: Intact (left) and partially degraded (right) RNA (1µl) on Bioanalyzer (nano
chip). The latter is still suitable to detect major differences in gene expression, but is far
from ideal!
cDNA synthesis
• Use 1-2 µg of total RNA (enough for 150-200 PCR reactions!) in a reaction of 25 µl
• Use random primers (we have a mix of four primers: NNNNNG/ NNNNNA/
NNNNNT/ NNNNNC)
• Denature RNA and primers (~25 pmol per µg total RNA) at 70 °C for 5-10 min (or at
95 °C for 2 min), and immediately transfer to ice-water.
• Mix components for cDNA-reaction at RT (per reaction, and calculate for 0.5* additional
reaction for >2 samples):
5µl 5x buffer
2.5 µl 0.1 M DTT (DTT is already present in the Promega buffer!)
1.25 µl 10 mM 4dNTP
0.75 µl RNAsin/RNAguard
xx µl H2O (to make total reaction 25µl)
1 µl MMLV (add MMLV just prior to adding the mix to the RNA, and mix well!).
*any RT is ok, we use normal MMLV and sometimes RNAse H- MMLV (both Promega),
MMLVH- is 5x more expensive and not necessary for cDNA used in qPCR, because the
amplicons are only 70-90 nts!
• Put RNA/random primer-mix at RT, and add cDNA-mix.
• Incubate for 1 h at 37° C (or on 42° when using the H- RT)
DO NOT add the “usual” 0.5 µl after 30 min, it hampers the PCR reaction and doesn’t
give a higher yield of cDNA, in addition this turns all reactions 50% more expensive,
which is a significant upregulation!
4
Protocol to prepare RNA for cDNA synthesis followed by realtime quantitative PCR
• Precipitate cDNA with 0.1 volume 3M NaAc pH 5.5, and 2.5-3 volumes of EtOH (and
glycogen or linear acrylamide when desired) on ice for 15-30 min (this helps to get rid of
contaminants that hamper the PCR reaction)
• Spin 14,000 rpm for 30 min at 4 °C
• Wash pellet with 75% EtOH, and resuspend in 25 µl
• In a 10 µl qPCR reaction use 0.05-0.1 µl of this cDNA (from ≥ 0.25 µl we usually see
inhibition of qPCR)
• Aliquot samples (calculate how much you’ll need for the # plates you want to run on 1
day and aliquot accordingly) and store at -20 or -80 °C (repeated freeze-thaw cycles
yields higher Ct-values).
Example: If you have 4 experimental & 4 control samples you could measure 17 primer
sets, in addition to 2 housekeeping genes in duplicate (=19 reactions), for these 8 samples
by running 2 plates on one day. Then you need 0.95 – 1.9 µl of cDNA per sample. Thus,
it is easiest to aliquot in a way that you only need to thaw 1 tube per day.
P ri mer1
P ri mer1
P ri mer2
Exp.1 Cont.1 Exp.1 Cont.1
P ri mer1
Exp.1 Cont.1 Exp.1 Cont.1
NTC
NTC
P ri mer1
P ri mer5
P ri mer1
P ri mer3
P ri mer5
P ri mer4
NTC
NTC
P ri mer1
P ri mer5
P ri mer5
P ri mer6
Exp.3 Cont.3 Exp.3 Cont.3
NTC
NTC
P ri mer1
P ri mer5
P ri mer7
P ri mer8
NTC
NTC
P ri mer3
P ri mer3
P ri mer5
P ri mer7
P ri mer7
Exp.3 Cont.3 Exp.3 Cont.3
P ri mer1
P ri mer5
P ri mer7
P ri mer7
P ri mer3
P ri mer3
P ri mer5
P ri mer7
Exp.2 Cont.2 Exp.2 Cont.2
P ri mer3
P ri mer3
P ri mer5
Exp.2 Cont.2 Exp.2 Cont.2
P ri mer1
P ri mer3
P ri mer3
P ri mer7
P ri mer7
P ri mer7
Exp.4 Cont.4 Exp.4 Cont.4
Exp.4 Cont.4 Exp.4 Cont.4
P ri mer2
P ri mer6
P ri mer2
P ri mer4
P ri mer4
Exp.1 Cont.1 Exp.1 Cont.1
P ri mer2
P ri mer2
P ri mer4
P ri mer4
P ri mer6
P ri mer8
P ri mer8
Exp.1 Cont.1 Exp.1 Cont.1
P ri mer6
P ri mer6
P ri mer8
P ri mer8
Exp.2 Cont.2 Exp.2 Cont.2
Exp.2 Cont.2 Exp.2 Cont.2
P ri mer2
P ri mer6
P ri mer2
P ri mer11 P ri mer12
NTC
NTC
HK 1
HK 2
NTC
NTC
P ri mer2
P ri mer6
P ri mer4
P ri mer6
P ri mer8
NTC
P ri mer8
Exp.4 Cont.4 Exp.4 Cont.4
P ri mer6
P ri mer10
Exp.3 Cont.3 Exp.3 Cont.3
P ri mer4
P ri mer4
NTC
Exp.3 Cont.3 Exp.3 Cont.3
P ri mer2
P ri mer4
P ri mer9
P ri mer8
P ri mer8
Exp.4 Cont.4 Exp.4 Cont.4
HK 1
HK 1
Exp.1 Cont.1
HK 1
HK 1
Exp.2 Cont.2
HK 1
HK 1
Exp.3 Cont.3
HK 1
HK 1
Exp.4 Cont.4
HK 2
HK 2
Exp.1 Cont.1
HK 2
HK 2
Exp.2 Cont.2
HK 2
HK 2
Exp.3 Cont.3
HK 2
HK 2
Exp.4 Cont.4
P ri mer9
P ri mer9
P ri mer11 P ri mer11 P ri mer13 P ri mer13 P ri mer15 P ri mer15 P ri mer13 P ri mer14
Exp.1 Cont.1 Exp.1 Cont.1
P ri mer9
P ri mer9
P ri mer9
P ri mer9
Exp.2 Cont.2 Exp.2 Cont.2
NTC
P ri mer11 P ri mer11 P ri mer13 P ri mer13 P ri mer15 P ri mer15 P ri mer17
Exp.3 Cont.3 Exp.3 Cont.3
P ri mer9
NTC
Exp.3 Cont.3 Exp.3 Cont.3
P ri mer11 P ri mer11 P ri mer13 P ri mer13 P ri mer15 P ri mer15
Exp.4 Cont.4 Exp.4 Cont.4
Exp.4 Cont.4 Exp.4 Cont.4
NTC
NTC
HK 1
NTC
HK 2
NTC
P ri mer10 P ri mer10 P ri mer12 P ri mer12 P ri mer14 P ri mer14 P ri mer16 P ri mer16 P ri mer17 P ri mer17
Exp.1 Cont.1 Exp.1 Cont.1
Exp.1 Cont.1 Exp.1 Cont.1 Exp.1 Cont.1
P ri mer10 P ri mer10 P ri mer12 P ri mer12 P ri mer14 P ri mer14 P ri mer16 P ri mer16 P ri mer17 P ri mer17
Exp.2 Cont.2 Exp.2 Cont.2
Exp.2 Cont.2 Exp.2 Cont.2 Exp.2 Cont.2
P ri mer10 P ri mer10 P ri mer12 P ri mer12 P ri mer14 P ri mer14 P ri mer16 P ri mer16 P ri mer17 P ri mer17
Exp.3 Cont.3 Exp.3 Cont.3
Exp.3 Cont.3 Exp.3 Cont.3 Exp.3 Cont.3
P ri mer10 P ri mer10 P ri mer12 P ri mer12 P ri mer14 P ri mer14 P ri mer16 P ri mer16 P ri mer17 P ri mer17
Exp.4 Cont.4 Exp.4 Cont.4
Exp.4 Cont.4 Exp.4 Cont.4 Exp.4 Cont.4
Figure 3. Example of plate set-up for 8 samples and 17 primer-sets (and two house
keeping genes in duplicate) using 2 plates to be run on one day.
• Always use filtertips when pipeting in the 2x SYBR mix (risk of contamination); do
NOT use your own pipets for pipeting in this solution (especially not when you are
performing cloning-steps (DNA digests, DNA preps) with these because this is a huge
risk of contamination!)
• Use the automatic pipet; this saves (filter)-tips and your muscles, in addition your
results become more reproducible
5
NTC
P ri mer11 P ri mer11 P ri mer13 P ri mer13 P ri mer15 P ri mer15 P ri mer15 P ri mer16
Exp.2 Cont.2 Exp.2 Cont.2
P ri mer9
Exp.1 Cont.1 Exp.1 Cont.1
HK 1
HK 1
Exp.1 Cont.1
HK 1
HK 1
Exp.2 Cont.2
HK 1
HK 1
Exp.3 Cont.3
HK 1
HK 1
Exp.4 Cont.4
HK 2
HK 2
Exp.1 Cont.1
HK 2
HK 2
Exp.2 Cont.2
HK 2
HK 2
Exp.3 Cont.3
HK 2
HK 2
Exp.4 Cont.4
Protocol to prepare RNA for cDNA synthesis followed by realtime quantitative PCR
•BUT, be aware of contamination (otherwise every well is very reproducible compared to
the other wells!), e.g. pipet primers to left side of the well, and the cDNA solution to the
right side.
cDNA-mi x (7µl)
prim er (3µl)
Figure 4. Order and place to pipet qPCR solutions.
First, the primer-mix is pipetted on the left side of
the tube (do NOT touch right side!!!), then the
cDNA-mix is pipetted at the top of the rim at the
right side. Be very careful not to touch the left
upper rim of the well with your tip, place your pipet
in the right angle!
• Spin tubes with cDNA and primers to prevent aerosol formation (and contamination).
Primers are stored at 4 °C, do not freeze these low concentrations.
• Wear gloves at all time, several primers are also human-specific and recognize DNA on
hands etc.
• Work in a hood or at least in a lab that is different from the one where DNA-work
(cloning/PCRing) occurs.
A typical reaction consists of:
5µl 2x SYBR-mix
• 1.9 µl H2O
• 0.1 µl cDNA/H2O
• 3 µl primermix (1 pmol/µl of sense and antisense primers)
For the example mentioned above (see Figure 3; 19 reactions per cDNA sample and 21
reactions for the NTC), there are 173 reactions. But, since you have to make separate
tubes for each sample, it is best to calculate an additional 0.5 reaction per sample
(cDNA+NTC) and 1 additional reaction for pipeting errors, meaning that there will be an
additional 5.5 reactions, so 178.5 reactions, which we’ll round up to 179.
6
Protocol to prepare RNA for cDNA synthesis followed by realtime quantitative PCR
• First mix for these 179 reactions (samples+NTC) the 2x SYBRmix and H2O (take care,
the SYBRmix is soapy, thus pipet slowly and do not fill the tip to the maximum volume
of the pipet).
• Divide this mixture over 9 fresh&clean tubes (8*cDNA+1*NTC). This means 8 tubes
with 19.5*5.9 µl SYBR/H2O-mix, and 1 tube with 21.5*5.9 µl SYBR/H2O-mix. Then,
close the 2xSYBRmix and put it away (do NOT leave it open while pipeting cDNA!!!).
• To each tube add the cDNA or H2O; 19.5*0.1 µl cDNA or 21.5*0.1 µl H2O. Mix well,
but be aware not to generate too much bubbles, also spin at low g (~5000g), because
spinning harder may cause SYBR to precipitate from the solution.
• Before opening and dividing the primersmixes I prefer to change my gloves to prevent
contamination of these primer-dilutions.
• Add the primermixes (3 µl) to the plate (using the automatic pipet according to Figure
4).
• Add the SYBR/H2O/cDNA (or- SYBR/H2O/H2O)-mix (7 µl) to the plate (using the
automatic pipet according to Figure 4). For small volumes always use the 40µl-extension
of the automatic pipet, because the 200µl-extension takes up a large “dead” volume. This
“dead” volume has to put back in the tube after pipetting the first plate, because otherwise
you may run short in the second plate.
• Apply cover on the plate using your gloves (do NOT use bare hands because at this time
your hands are covered with powder from the gloves!).
• Spin plate at 1000 rpm for ~ 20 sec to get the samples to the bottom of the plate, and to
remove possible air bubbles.
7