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-PCRfor 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 around150 mgof sample (10 day old Arabidopsis seedlings) in liquid nitrogen and grind thoroughly witha mortar and pestle.
Note: The RNA in the plant sample is not protected after harvesting until the sample is flash frozenin liquid nitrogen. Frozen tissue should not be allowed to thaw during handling. Therelevant 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 thatβ-ME is added to Buffer RLT before use.
4. Pipet the lysate directly onto a QIAshredder spin column (lilac) placed in 2 ml collectiontube, and centrifuge for 2 min at maximum speed. Carefully transfer the supernatantof the flow-through fraction to a newmicrocentrifuge tube withoutdisturbing the cell debris pellet in the collection tube. Use only this supernatant insubsequent steps.
Note: Centrifugation through the QIAshredder spin columnremoves cell debris and simultaneously homogenizes the lysate. While most of thecell debris is retained on the QIAshredder spin column, a very small amount of celldebris 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, toan RNeasy mini column (pink) placed in a 2 ml collection tube (supplied). Close thetube 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 centrifugefor 15 s to wash the column. Discard the flow-throughand collection tube.
8. Transfer the RNeasy column into a new 2 ml collection tube (supplied). Pipet 500 µlBuffer 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 andcentrifuge 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 pipet50 µl RNase-free water directly onto the RNeasy silica-gel membrane. Close thetube 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 byspectrophotometry (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 avolume 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 / Volume/reaction / Master mix / Final concentrationRNase-free water / µl / -
5X RT-PCR buffer / 10 µl / 1X
dNTP mix / 2 µl / 400 µM of each dNTP
AtAGP19 primers (10 µM of each primer) / 6µl / 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 / µl / 1 µg/reaction
Total volume / 50 µl / -
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 usinga 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 requirecofactors to function. Since RNases are difficult to inactivate and even minute amounts aresufficient to destroy RNA, do not use any plasticware or glassware without first eliminatingpossible RNase contamination. Great care should be taken to avoid inadvertentlyintroducing RNases into the RNA sample during or after the isolation procedure. In orderto create and maintain an RNase-free environment, the following precautions must betaken during pretreatment and use of disposable and non-disposable vessels and solutionswhile 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 sourcesof RNase contamination. Always wear latex or vinyl gloves while handling reagents andRNA samples to prevent RNase contamination from the surface of the skin or from dustylaboratory 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 byRNase-free water.
Glassware
Glassware should be treated before use to ensure that it is RNase-free. Glassware usedfor RNA work should be cleaned with a detergent, thoroughly rinsed, and oven baked at240°C for four or more hours (or overnight, if more convenient) before use. Autoclavingalone will not fully inactivate many RNases. Alternatively, glassware can be treated withDEPC (diethyl pyrocarbonate). Fill glassware with 0.1% DEPC (0.1% in water), allow tostand overnight (12 hours) at 37°C, and then autoclave or heat to 100°C for 15 minutesto 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% toinactivate 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 thesolution to be treated and shake vigorously to bring the DEPC into solution. Let thesolution incubate for 12 hours at 37°C. Autoclave for 15 minutes to remove any trace ofDEPC. DEPC will react with primary amines and cannot be used directly to treat Trisbuffers. DEPC is highly unstable in the presence of Tris buffers and decomposes rapidlyinto ethanol and CO2. When preparing Tris buffers, treat water with DEPC first, and thendissolve Tris to make the appropriate buffer. Trace amounts of DEPC will modify purineresidues in RNA by carboxymethylation. Carboxymethylated RNA is translated with verylow efficiency in cell-free systems. However, its ability to form DNA:RNA or RNA:RNAhybrids is not seriously affected unless a large fraction of the purine residues have beenmodified. Residual DEPC must always be eliminated from solutions or vessels byautoclaving or heating to 100°C for 15 minutes.
Appendix B: Storage, Quantitation, and Determinationof Quality of Total RNA
Storage of RNA
Purified RNA may be stored at –20°C or –70°C in water. Under these conditions, nodegradation 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 greaterthan 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, ifit 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 givesan estimate of RNA purity.When measuring RNA samples, be certain that cuvettes are RNase-free, especially if the RNAis to be recovered after spectrophotometry. This can be accomplished by washing cuvetteswith 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 thepurity 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 pHand the resulting A260/A280 ratio can vary greatly. Lower pH results in a lower A260/A280ratio and reduced sensitivity to protein contamination. For accurate values, we recommendmeasuring 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 spectrophotometerwith the same solution.
For determination of RNA concentration, however, we still recommend dilution of the samplein water since the relationship between absorbance and concentration (A260 reading of1 = 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 ofDNA, even when it is not visible on an agarose gel. To prevent any interference by DNAin RT-PCR applications,Qiagen recommends designing primers that anneal at intron splicejunctions so that genomic DNA will not be amplified. Alternatively, DNA contaminationcan be detected on agarose gels following RT-PCR by performing control experiments inwhich no reverse transcriptase is added prior to the PCR step or by using intron-spanningprimers. For sensitive applications, such as differential display, or if it is not practical touse splice-junction primers, DNase digestion of the purified RNA with RNase-free DNaseis recommended.
A protocol for optional on-column DNase digestion using the RNase-Free DNase Set isprovided above. The DNase is efficiently washed away in thesubsequent wash steps. Alternatively, after the RNeasy procedure, the eluate containingthe RNA can be treated with DNase. The RNA can then be repurified with the RNeasycleanup protocol, or after heat inactivation of the DNase, the RNA can be useddirectly in downstream applications.
Integrity of RNA
The integrity and size distribution of total RNA purified with RNeasy Kits can be checkedby denaturing agarose gel electrophoresis and ethidium bromide staining. Therespective 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 thatof 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 sufferedmajor 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 andspecific RT-PCR using any RNA. The kit contains optimized components that allowboth reverse transcription and PCR amplification to take place in what is commonlyreferred to as a “one-step” reaction.
QIAGEN OneStep RT-PCR Enzyme Mix
The QIAGEN OneStep RT-PCR Enzyme Mix contains a specially formulated enzyme blendfor both reverse transcription and PCR amplification.
• Omniscript and Sensiscript Reverse Transcriptases are included in the QIAGENOneStep RT-PCR Enzyme Mix and provide highly efficient and specific reversetranscription. Both reverse transcriptases exhibit a higher affinity for RNA, facilitatingtranscription through secondary structures that inhibit other reverse transcriptases.Omniscript Reverse Transcriptase is specially designed for reverse transcription ofRNA amounts greater than 50 ng, and Sensiscript Reverse Transcriptase is optimizedfor use with very small amounts of RNA (<50 ng). This special enzyme combinationin the QIAGEN OneStep RT-PCR Enzyme Mix provides highly efficient andsensitive reverse transcription of any RNA quantity from 1 pg to 2 µg.
• HotStarTaq DNA Polymerase included in the QIAGEN OneStep RT-PCR EnzymeMix provides hot-start PCR for highly specific amplification. During reverse transcription,HotStarTaq DNA Polymerase is completely inactive and does notinterfere with the reverse-transcriptase reaction. After reverse transcription byOmniscript and Sensiscript Reverse Transcriptases, reactions are heated to 95°C for15 min to activate HotStarTaq DNA Polymerase and to simultaneously inactivate thereverse transcriptases. This hot-start procedure using HotStarTaq DNA Polymeraseeliminates extension from nonspecifically annealed primers and primer–dimers in thefirst cycle ensuring highly specific and reproducible PCR.
Although all of the enzymes are present in the reaction mix, the use of HotStarTaq DNAPolymerase ensures the temporal separation of reverse transcription and PCR allowingboth processes to be performed sequentially in a single tube. Only one reaction mix needsto 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 andspecific amplification.
• The unique buffer composition allows reverse transcription to be performed at hightemperatures (50°C). This high reaction temperature improves the efficiency of thereverse-transcriptase reaction by disrupting secondary structures and is particularlyimportant for one-step RT-PCR performed with limiting template RNA amounts.
• It has been reported that one-step RT-PCR may exhibit reduced PCR efficiencycompared to two-step RT-PCR. The combination of QIAGEN enzymes and the uniqueformulation of the QIAGEN OneStep RT-PCR Buffer ensures high PCR efficiency ina one-step RT-PCR.
• The buffer contains the same balanced combination of KCl and (NH4)2SO4 included inQIAGEN PCR Buffer. This formulation enables specific primer annealing over awider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers.The need for optimization of RT-PCR by varying the annealingtemperature 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 semiquantitativeor relative RT-PCR. Typically, the signal from the RT-PCR product is normalizedto the signal from an internal control included in all samples and amplified at the sametime 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 asinternal controls because they are abundantly expressed at relatively constant rates in mostcells. However, the internal control transcript is usually more abundant than the transcriptunder study. This difference in abundance can lead to preferential amplification of theinternal 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 surethat the products can be easily distinguished on an agarose gel.
• Determine RT-PCR conditions that are suitable for both amplicons by varyingtemplate amount, number of cycles, annealing temperature, and extensiontime.
• Initially, try primer concentrations of 0.6 µM for the target transcript and 0.3 µMfor the internal control transcript.
• If the yield of internal standard greatly exceeds that of the specific target usingthe concentrations given above, reduce the internal-control primer concentrationin steps of 0.05–0.1 µM. The optimal primer concentration for the internalcontrol depends on the relative abundance and efficiency of amplification ofthe control and target transcripts. Control transcripts may be much more highlyexpressed 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 thetarget transcript.
General guidelines for standard RT-PCR primers