RNA extraction/RT-PCR (reverse transcriptase-polymerase chain reaction) Lab
Before you start, carefully read all the following steps, notes and appendixes (adapted
from QIAGEN RNeasy Mini Handbook and OneStep RT-PCR Kit Handbook with
modifications made by the TA). The kits to be used are from QIAGEN.
Exercise 1. RNA extraction
Make sure you know how to handle RNA. Be careful to retain a RNase-free environment. If
you get no RNA, you cannot do a RT-PCR for your lab report!
Steps 1 to 11 are performed at room temperature. During the procedure, work quickly.
Centrifuge at full speed in a bench-top centrifuge.
1. Add 4.5 µl β-Mercaptoethanol (β-ME) to 450 µl Buffer RLT in a fume hood. Weigh
the microcentrifuge tube and record the weight.
2. Place around 150 mg of sample (10 day old Arabidopsis seedlings) in liquid nitrogen
and grind thoroughly with a mortar and pestle.
Note: The RNA in the plant sample is not protected after harvesting until the sample is flash frozen in
liquid nitrogen. Frozen tissue should not be allowed to thaw during handling. The relevant procedures
should be carried out as quickly as possible.
3. Add up to 100 mg tissue powder to the prepared Buffer RLT. Vortex vigorously.
Note: Ensure that β-ME is added to Buffer RLT before use.
4. Pipet the lysate directly onto a QIA shredder spin column (lilac) placed in 2 ml
collection tube, and centrifuge for 2 min at maximum speed. Carefully transfer the
supernatant of the flow-through fraction to a new microcentrifuge tube without
disturbing the cell debris pellet in the collection tube. Use only this supernatant in
subsequent steps.
Note: Centrifugation through the QIA shredder spin column removes cell debris and simultaneously
homogenizes the lysate. While most of the cell debris is retained on the QIA shredder spin column, a very
small amount of cell debris will pass through and form a pellet in the collection tube.
5. Add 0.5 volume (usually 250 µl) ethanol (96–100%) to the cleared lysate, and mix
immediately by pipetting. Do not centrifuge. Continue without delay.
6. Apply sample, including any precipitate that may have formed, to an RNeasy mini
column (pink) placed in a 2 ml collection tube (supplied). Close the tube gently, and
centrifuge for 15 s. Discard the flow-through.
Reuse the collection tube in step 7. Discard the flow-through after each centrifugation step.
7. Add 700 µl Buffer RW1 to the RNeasy column. Close the tube gently, and centrifuge
for 15 s to wash the column. Discard the flow-through and collection tube.
8. Transfer the RNeasy column into a new 2 ml collection tube (supplied). Pipet 500 µl
Buffer RPE onto the RNeasy column. Close the tube gently, and centrifuge for 15 s to
wash the column. Discard the flow-through.
Reuse the collection tube in step 9.
9. Add another 500 µl Buffer RPE to the RNeasy column. Close the tube gently, and
centrifuge for 1 min to dry the RNeasy silica-gel membrane.
10. Place the RNeasy column in a new microcentrifuge tube and centrifuge for 1 min.
Note: It is important to dry the RNeasy silica-gel membrane since residual ethanol may
interfere with downstream reactions. This centrifugation ensures that no ethanol is carried
over during elution.
11. To elute, transfer the RNeasy column to a new 1.5 ml collection tube. Then pipet 50
µl RNase-free water directly onto the RNeasy silica-gel membrane. Close the tube gently,
and centrifuge for 1 min to elute.
12. Keep the RNA on ice. Dilute 1 µl RNA into 49 µl water and determine the RNA
concentration by spectrophotometry (see appendix below).
Exercise 2. RT-PCR
QIAGEN OneStep RT-PCR Kit allows reverse transcription and PCR are carried out
sequentially in the same tube. Two pairs of primers will be used: one pair is for AtAGP19
(At1g68725), the gene of interest; and the other pair is for actin, which serves as an internal
control.
1. Each group will prepare a master mix with a volume 10% greater than that required
for the total number of reactions to be performed (For example, for 3 reactions, prepare
3.3 reactions of master mix). You will learn to calculate the volume of each component
required for your group’s master mix. Fill in the following table.
Component
RNase-free water
5X RT-PCR buffer
Volume/reaction
µl
Master mix
Final concentration
-
10 µl
1X
dNTP mix
2 µl
400 µM of each dNTP
AtAGP19 primers (10
µM of each primer)
6 µl
0.6 µM of each primer
Actin primers (10 µM
of each primer)
2 µl
0.2 µM of each primer
Enzyme
2 µl
-
Template RNA
Total volume
µl
50 µl
1 µg/reaction
-
Note: Optimal primer concentrations used for RT-PCR and PCR are different as seen above for the higher
concentrations of primers for the gene of interest and the internal control in the above RT-PCR table.
2. Set up the reactions on ice according to the above table. Add the RNA last.
3. Preheat the thermal cycler to 50°C and then place the samples into the cycler.
4. Perform the following cycles: 50°C 30 min (reverse transcription); 95°C 15 min
(initial PCR activation and reverse transcriptase inactivation); 30 cycles of (94°C 45 sec,
50°C 45 sec and 72°C 1 min); 72°C 10 min; and finally hold at 4°C.
5. After RT-PCR, products will be run on a 1.2% agarose gel in TAE buffer using a 100
bp ladder as the molecular size marker (New England Biolabs). Make sure you know
the origin (i.e., DNA or RNA template) of each band amplified in this RT-PCR.
Appendix A: General Remarks on Handling RNA
Ribonucleases (RNases) are very stable and active enzymes that generally do not require
cofactors to function. Since RNases are difficult to inactivate and even minute amounts are
sufficient to destroy RNA, do not use any plasticware or glassware without first eliminating
possible RNase contamination. Great care should be taken to avoid inadvertently introducing
RNases into the RNA sample during or after the isolation procedure. In order to create and
maintain an RNase-free environment, the following precautions must be taken during
pretreatment and use of disposable and non-disposable vessels and solutions while working
with RNA.
General handling
Proper microbiological, aseptic technique should always be used when working with RNA.
Hands and dust particles may carry bacteria and molds and are the most common sources of
RNase contamination. Always wear latex or vinyl gloves while handling reagents and RNA
samples to prevent RNase contamination from the surface of the skin or from dusty
laboratory equipment. Change gloves frequently and keep tubes closed whenever possible.
Keep isolated RNA on ice when aliquots are pipetted for downstream applications.
Disposable plasticware
The use of sterile, disposable polypropylene tubes is recommended throughout the procedure.
These tubes are generally RNase-free and do not require pretreatment to inactivate RNases.
Non-disposable plasticware
Non-disposable plasticware should be treated before use to ensure that it is RNase-free.
Plasticware should be thoroughly rinsed with 0.1 M NaOH, 1 mM EDTA followed by
RNase-free water.
Glassware
Glassware should be treated before use to ensure that it is RNase-free. Glassware used for
RNA work should be cleaned with a detergent, thoroughly rinsed, and oven baked at 240°C
for four or more hours (or overnight, if more convenient) before use. Autoclaving alone will
not fully inactivate many RNases. Alternatively, glassware can be treated with DEPC (diethyl
pyrocarbonate). Fill glassware with 0.1% DEPC (0.1% in water), allow to stand overnight (12
hours) at 37°C, and then autoclave or heat to 100°C for 15 minutes to eliminate residual
DEPC.
DEPC is a suspected carcinogen and should be handled with great care. Wear gloves and
use a fume hood
Solutions
Solutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is a strong,
but not absolute, inhibitor of RNases. It is commonly used at a concentration of 0.1% to
inactivate RNases on glass or plasticware or to create RNase-free solutions and water. DEPC
inactivates RNases by covalent modification. Add 0.1 ml DEPC to 100 ml of the solution to
be treated and shake vigorously to bring the DEPC into solution. Let the solution incubate for
12 hours at 37°C. Autoclave for 15 minutes to remove any trace of DEPC. DEPC will react
with primary amines and cannot be used directly to treat Tris buffers. DEPC is highly
unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO2. When
preparing Tris buffers, treat water with DEPC first, and then dissolve Tris to make the
appropriate buffer. Trace amounts of DEPC will modify purine residues in RNA by
carboxymethylation. Carboxymethylated RNA is translated with very low efficiency in
cell-free systems. However, its ability to form DNA:RNA or RNA:RNA hybrids is not
seriously affected unless a large fraction of the purine residues have been modified. Residual
DEPC must always be eliminated from solutions or vessels by autoclaving or heating to
100°C for 15 minutes.
Appendix B: Storage, Quantitation, and Determination of Quality of Total RNA
Storage of RNA
Purified RNA may be stored at –20°C or –70°C in water. Under these conditions, no
degradation of RNA is detectable after 1 year.
Quantitation of RNA
The concentration of RNA should be determined by measuring the absorbance at 260 nm
(A260) in a spectrophotometer. To ensure significance, readings should be greater than 0.15.
An absorbance of 1 unit at 260 nm corresponds to 40 µg of RNA per ml. This relation is valid
only for measurements in water. Therefore, if it is necessary to dilute the RNA sample, this
should be done in water. As discussed below (see “Purity of RNA”), the ratio between the
absorbance values at 260 and 280 nm gives an estimate of RNA purity. When measuring
RNA samples, be certain that cuvettes are RNase-free, especially if the RNA is to be
recovered after spectrophotometry. This can be accomplished by washing cuvettes with 0.1M
NaOH, 1 mM EDTA followed by washing with RNase-free water. Use the buffer in which
the RNA is diluted to zero the spectrophotometer.
Purity of RNA
The ratio of the readings at 260 nm and 280 nm (A260/A280) provides an estimate of the
purity of RNA with respect to contaminants that absorb in the UV, such as protein. However,
the A260/A280 ratio is influenced considerably by pH. Since water is not buffered, the pH
and the resulting A260/A280 ratio can vary greatly. Lower pH results in a lower A260/A280
ratio and reduced sensitivity to protein contamination. For accurate values, we recommend
measuring absorbance in 10 mM Tris·Cl, pH 7.5. Pure RNA has an A260/A280 ratio of
1.9–2.1 in 10 mM Tris·Cl, pH 7.5. Always be sure to calibrate the spectrophotometer with the
same solution.
For determination of RNA concentration, however, we still recommend dilution of the
sample in water since the relationship between absorbance and concentration (A260 reading
of 1 = 40 µg/ml RNA) is based on an extinction coefficient calculated for RNA in water.
DNA contamination
No currently available purification method can guarantee that RNA is completely free of
DNA, even when it is not visible on an agarose gel. To prevent any interference by DNA in
RT-PCR applications, Qiagen recommends designing primers that anneal at intron splice
junctions so that genomic DNA will not be amplified. Alternatively, DNA contamination can
be detected on agarose gels following RT-PCR by performing control experiments in which
no reverse transcriptase is added prior to the PCR step or by using intron-spanning primers.
For sensitive applications, such as differential display, or if it is not practical to use
splice-junction primers, DNase digestion of the purified RNA with RNase-free DNase is
recommended.
A protocol for optional on-column DNase digestion using the RNase-Free DNase Set is
provided above. The DNase is efficiently washed away in the subsequent wash steps.
Alternatively, after the RNeasy procedure, the eluate containing the RNA can be treated with
DNase. The RNA can then be repurified with the RNeasy cleanup protocol, or after heat
inactivation of the DNase, the RNA can be used directly in downstream applications.
Integrity of RNA
The integrity and size distribution of total RNA purified with RNeasy Kits can be checked by
denaturing agarose gel electrophoresis and ethidium bromide staining. The respective
ribosomal bands (Table 1) should appear as sharp bands on the stained gel.
28S ribosomal RNA bands should be present with an intensity that is approximately twice
that of the 18S RNA band (Figure 1). If the ribosomal bands in a given lane are not sharp, but
appear as a smear of smaller sized RNAs, it is likely that the RNA sample suffered major
degradation during preparation.
Table 1. Size of ribosomal RNAs from various sources
Appendix C: Introduction to the QIAGEN OneStep RT-PCR Kit and RT-PCR
The QIAGEN OneStep RT-PCR Kit provides a convenient format for highly efficient and
specific RT-PCR using any RNA. The kit contains optimized components that allow both
reverse transcription and PCR amplification to take place in what is commonly referred to as
a “one-step” reaction.
QIAGEN OneStep RT-PCR Enzyme Mix
The QIAGEN OneStep RT-PCR Enzyme Mix contains a specially formulated enzyme blend
for both reverse transcription and PCR amplification.
• Omniscript and Sensiscript Reverse Transcriptases are included in the QIAGEN OneStep
RT-PCR Enzyme Mix and provide highly efficient and specific reverse transcription. Both
reverse transcriptases exhibit a higher affinity for RNA, facilitating transcription through
secondary structures that inhibit other reverse transcriptases. Omniscript Reverse
Transcriptase is specially designed for reverse transcription of RNA amounts greater than 50
ng, and Sensiscript Reverse Transcriptase is optimized for use with very small amounts of
RNA (<50 ng). This special enzyme combination in the QIAGEN OneStep RT-PCR Enzyme
Mix provides highly efficient and sensitive reverse transcription of any RNA quantity from 1
pg to 2 µg.
• HotStarTaq DNA Polymerase included in the QIAGEN OneStep RT-PCR Enzyme Mix
provides hot-start PCR for highly specific amplification. During reverse transcription,
HotStarTaq DNA Polymerase is completely inactive and does not interfere with the
reverse-transcriptase reaction. After reverse transcription by Omniscript and Sensiscript
Reverse Transcriptases, reactions are heated to 95°C for 15 min to activate HotStarTaq DNA
Polymerase and to simultaneously inactivate the reverse transcriptases. This hot-start
procedure using HotStarTaq DNA Polymerase eliminates extension from nonspecifically
annealed primers and primer–dimers in the first cycle ensuring highly specific and
reproducible PCR.
Although all of the enzymes are present in the reaction mix, the use of HotStarTaq DNA
Polymerase ensures the temporal separation of reverse transcription and PCR allowing both
processes to be performed sequentially in a single tube. Only one reaction mix needs to be set
up: no additional reagents are added after the reaction starts.
QIAGEN OneStep RT-PCR Buffer
QIAGEN OneStep RT-PCR Buffer is designed to enable both efficient reverse transcription
and specific amplification.
• The unique buffer composition allows reverse transcription to be performed at high
temperatures (50°C). This high reaction temperature improves the efficiency of the
reverse-transcriptase reaction by disrupting secondary structures and is particularly important
for one-step RT-PCR performed with limiting template RNA amounts.
• It has been reported that one-step RT-PCR may exhibit reduced PCR efficiency compared
to two-step RT-PCR. The combination of QIAGEN enzymes and the unique formulation of
the QIAGEN OneStep RT-PCR Buffer ensures high PCR efficiency in a one-step RT-PCR.
• The buffer contains the same balanced combination of KCl and (NH4)2SO4 included in
QIAGEN PCR Buffer. This formulation enables specific primer annealing over a wider range
of annealing temperatures and Mg2+ concentrations than conventional PCR buffers. The need
for optimization of RT-PCR by varying the annealing temperature or the Mg2+ concentration
is therefore minimized.
Co-amplification of an internal control
The relative abundance of a transcript in different samples can be estimated by
semiquantitative or relative RT-PCR. Typically, the signal from the RT-PCR product is
normalized to the signal from an internal control included in all samples and amplified at the
same time as the target. The normalized data from different samples can then be compared.
Transcripts of housekeeping genes such as GAPDH or actin are frequently chosen as internal
controls because they are abundantly expressed at relatively constant rates in most cells.
However, the internal control transcript is usually more abundant than the transcript under
study. This difference in abundance can lead to preferential amplification of the internal
control and, in some cases, prevent amplification of the target RT-PCR product. Often, such
problems can be overcome by reducing the internal-control primer concentration. The
following guidelines may be helpful in developing co-amplification conditions:
• Choose similar amplicon sizes for the target and the internal control but be sure that the
products can be easily distinguished on an agarose gel.
• Determine RT-PCR conditions that are suitable for both amplicons by varying template
amount, number of cycles, annealing temperature, and extension time.
• Initially, try primer concentrations of 0.6 µM for the target transcript and 0.3 µM for the
internal control transcript.
• If the yield of internal standard greatly exceeds that of the specific target using the
concentrations given above, reduce the internal-control primer concentration in steps of
0.05–0.1 µM. The optimal primer concentration for the internal control depends on the
relative abundance and efficiency of amplification of the control and target transcripts.
Control transcripts may be much more highly expressed than the target transcript. If the
difference in abundance is too great, then RT-PCR of the internal control may interfere with
the amplification of the target transcript.
General guidelines for standard RT-PCR primers