Preparation Of Isotopic Or Nonisotopic Dnarna Probes

Protocol A. Preparation of DNA Probes

Detailed protocols are described in Chapter 7.

Protocol B. Preparation of RNA Probes by Transcription in Vitro

A number of plasmid vectors have been developed for subcloning cDNA. These vectors have polycloning sites downstream from powerful bacteriophage promoters, SP6, T7 or T3 in the vector. The cDNA of interest can be cloned at the polycloning site between promoters SP6 and T7 or T3, forming a recombinant plasmid. The cDNA inserted can be transcribed in vitro into single-strand sense RNA or antisense RNA from a linear plasmid DNA with promoters SP6, T7 or T3. During the process of in vitro transcription, one of the rNTPs is radioactively labeled and can be

Water bottle with 500 ml water

Glass or plastic plate

5-8 cm thickness of paper towels

Membrane filter RNA agarose gel

Support Tray

Water bottle with 500 ml water

Glass or plastic plate

5-8 cm thickness of paper towels

Two sheets of 3MM Whatman paper

Long sheet of 3MM Whatman paper as a wick

10X SSC used as transfer buffer

FIGURE 10.2 Standard assembly for upward capillary transfer of RNAs from an agarose gel onto a nylon membrane filter.

Transfer buffer

Glass or plastic plate

Long sheet of 3MM Whatman paper as a wick

Two sheets of wet 3MM Whatman paper

Transfer buffer

Transfer buffer

8-10 cm thickness of paper towels

FIGURE 10.3 Standard assembly of downward capillary transfer of RNAs from an agarose gel onto a nylon membrane filter.

Transfer buffer

Two sheets of wet 3MM Whatman paper Four sheets of dry 3MM Whatman paper

Saran Wrap film

8-10 cm thickness of paper towels

FIGURE 10.3 Standard assembly of downward capillary transfer of RNAs from an agarose gel onto a nylon membrane filter.

incorporated into the RNAs. These are the labeled RNAs. The labeled RNA probes usually have a high specific activity and are much "hotter" than ssDNA probes. Compared with DNA labeling, the yield of the RNA probe is usually very high because the template can be repeatedly transcribed. RNA probes can be easily purified from a DNA template merely by using RNase-free DNAse I treatment. The greatest advantage of an RNA probe over a DNA probe is that the RNA probe can produce much stronger signals in a variety of different hybridization reactions.

1. Preparation of linear DNA template for transcription in vitro. The plasmid is linearized by an appropriate restriction enzyme in order to produce "run-off' transcripts. To make RNA transcripts from the DNA insert, recombinant plasmid DNA should be digested by an appropriate restriction enzyme that cuts at one site that is very close to one end of the insert.

a. On ice, set up a plasmid linearization reaction: Recombinant plasmid DNA (mg/ml), 5 mg Appropriate restriction enzyme 10X buffer, 2.5 ml Appropriate restriction enzyme, 3 m/mg DNA Add dd.H2O to a final volume of 25 ml.

b. Incubate at the appropriate temperature for 2 to 3 h.

c. Extract DNA with one volume of TE-saturated phenol/chloroform and mix well by vortexing. Centrifuge at 11,000 x g for 5 min at room temperature.

d. Carefully transfer the top, aqueous phase to a fresh tube and add one volume of chloroform:isoamyl alcohol (24:1). Mix well and centrifuge as described in the previous step.

e. Transfer the top, aqueous phase to a fresh tube. Add 0.1 volume of 2 M NaCl solution and two volumes of chilled 100% ethanol to the supernatant. Precipitate the DNA at -20°C for 30 min.

f. Centrifuge at 12,000 x g for 5 min, aspirate the supernatant and briefly rinse the pellet with 1 ml of 70% ethanol. Dry the DNA pellet for 10 min under vacuum and dissolve the linearized plasmid in 15 ml ddH2O.

g. Take 2 ml of the sample to measure the concentration of the DNA at A260 nm and A280 nm. Store the sample at -20°C until use.

2. Blunt the 3' overhang ends using the 3' ^ 5' exonuclease activity of Klenow DNA polymerase.

Note: Although this is optional, we recommend that the 3' protruding ends be converted into blunt ends because some of the RNA sequence is complementary to vector DNA. Therefore, enzymes such as Kpn I, Sac I, Pst I, bgl I, Sac II, Pvu I, Sfi and Sph I should not be used to linearize plasmid DNA for transcription in vitro.

3. Carry out synthesis of RNA via in vitro transcription.

a. Set up a reaction as follows: 5X transcription buffer, 8 ml 0.1 M DTT, 4 ml rRNasin ribonuclease inhibitor, 40 units Linearized template DNA (0.2 to 1.0 g/ml), 2 ml Add dd.H2O to a final volume of 15.2 ml.

b. Add Klenow DNA polymerase (5 u/mg DNA) to the reaction and incubate at 22°C for 15 to 20 min.

c. To the reaction, add the following components: Mixture of ATP, GTP, CTP, or UTP (2.5 mM each), 8 ml 120 mM UTP or CTP, 4.8 ml

SP6, T7, or T3 RNA polymerase (15 to 20 u/ml), 2 ml d. Incubate the reaction at 37 to 40°C for 1 h.

e. Remove DNA template using DNase I, using 1 u/mg DNA template. Incubate for 15 min at 37°C.

f. Extract RNA with one volume of TE-saturated phenol/chloroform. Mix well by vortexing for 1 min and centrifuging at 11,000 x g for 5 min at room temperature.

g. Carefully transfer the top, aqueous phase to a fresh tube and add one volume of chloroform:isoamyl alcohol (24:1). Mix well by vortexing and centrifuge at 11,000 x g for 5 min.

h. Carefully transfer the upper, aqueous phase to a fresh tube, add 0.5 volume of 7.5 ammonium acetate solution and 2.5 volumes of chilled 100% ethanol. Precipitate RNA at -80°C for 30 min.

i. Centrifuge at 12,000 x g for 10 min. Carefully discard the supernatant and briefly rinse the RNA with 1 ml of 70% ethanol and dry the pellet under vacuum for 15 min.

j. Dissolve the RNA probe in 20 to 50 ml of TE buffer and store at -20°C until use.

4. Calculate the percentage of incorporation and the specific activity of the

RNA probe.

a. Estimate the cpm used in the transcription reaction. For instance, if 50 mCi of NTP is used, the cpm is 50 x 2.2 x 106 cpm/mCi = 110 x 106 cpm in 40 ml reaction, or 2.8 x 106 cpm/ml.

b. Perform a TCA precipitation assay using 1:10 dilution in dd.H2O as described previously.

c. Calculate the percentage of incorporation: % incorporation = TCA precipitated cpm/total cpm x 100.

d. Calculate the specific activity of the probe. For example, if 1 ml of a 1:10 dilution is used for TCA precipitation, 10 x cpm precipitated = cpm/ml incorporated. In 40 ml of reaction, 40 x cpm/ml is the total cpm incorporated. If 50 mCi of labeled UTP at 400 mCi/nmol is used, then 50/400 = 0.125 nmol of UTP is added to the reaction mixture. If there is 100% incorporation and UTP represents 25% of the nucleotides in the RNA probe, 4 x 0.125 = 0.5 nmol of nucleotides is incorporated, and 0.5 x 330 ng/nmol = 165 ng of RNA is synthesized. Then, the total ng RNA probe = % incorporation x 165 ng. For example, if 1:10 dilution of the labeled RNA sample has 2.2 x 105 cpm, the total cpm incorporated is 10 x 2.2 x 105 cpm x 40 ml (total reaction) = 88 x 106 cpm. % incorporation = 88 x 106 cpm/110 x 106 cpm (50 mCi) = 80% total RNA synthesized = 165 ng x 0.80 = 132 ng RNA. The specific activity of the probe = 88 x 106 cpm/0.132 mg = 6.7 x 108 cpm/mg RNA

Note: An alternative is to synthesize RNA without using an isotope. The synthesized RNA can be labeled using nonradioactive approaches as described in Chapter 7.

Reagents Needed RNase-free DNase I 2 M NaCl solution 7.5 M Ammonium acetate solution Ethanol (100%, 70%) TE-Saturated phenol/chloroform Chloroform:isoamyl alcohol 24:1 (v/v) TE buffer

5X Transcription Buffer

200 mM Tris-HCl, pH 7.5 30 mM MgCl2 10 mM Spermidine 50 mM NaCl

NTPs Stock Solutions

10 mM ATP in dd.H2O, pH 7.0 10 mM GTP in dd.H2O, pH 7.0 10 mM UTP in dd.H2O, pH 7.0 10 mM CTP in dd.H2O, pH 7.0

Radioactive NTP Solution


The general procedures are the same as for DNA hybridization and detection, which are described in detail in Chapter 7.


The disadvantage of northern blot hybridization or of in situ hybridization of mRNA is the difficulty in obtaining a desired results when analyzing nonconstitutively expressed, cell- or tissue-specific genes with a low abundance of mRNAs or when limiting amounts of mRNA are available due to their degradation. These drawbacks, however, can be overcome by using quantitative PCR. The same amount of total RNAs or mRNAs from different samples is reverse transcribed into cDNAs under the same conditions. A particular cDNA of interest in total cDNAs in each of the samples is then amplified by PCR, using specific primers. An equal amount of the amplified cDNA from each sample is then analyzed by dot blotting or Southern blotting using the target sequence between primers as a probe. In this case, different amounts of mRNA expressed by a specific gene under different conditions will generate different amounts of cDNA. The different levels of gene expression can be detected with ease by comparing the hybridized signals of different samples. Meanwhile, a control mRNA (usually an appropriate housekeeping gene transcript) such as actin mRNA is reverse transcribed and amplified by PCR, using specific primers, under exactly the same conditions in order to monitor equivalent reverse transcription and an equivalent amplification by PCR.

1. Synthesize the first-strand cDNAs from the isolated total RNAs or mRNAs using reverse transcriptase and oligo(dT) as a primer. This is described in detail in Chapter 3.

2. Design primers for amplification of specific cDNA PCR. Design oligo-nucleic acid primers based on conserved amino acid sequences in two different regions of a specific cDNA. For instance, two oligonucleotide primers can be designed as follows for the two amino acid sequence regions of invertase, NDPNG and DPCEW.

Forward primer = 5' AAC(T)GAT(C)CCIAA(C)TGGI 3', derived from NDPNG

Reverse primer = 3' GGTGAGCGTCCCTAG 5', derived from DPCEW Note: I stands for the third position of the codon, which can be any of

TCAG. The primers can generate a product of554 base pairs. For amplification of actin cDNA, the primers can be designed as follows: Forward primer = 5' ATGGATGACGATATCGCTG 3' Reverse primer = 5' ATGAGGTAGTCTGTCAGGT 3' Note: These primers can generate a product of568 base pairs.

3. Carry out PCR amplification.

a. In a 0.5-ml microcentrifuge tube on ice, add the following items in the order listed for one sample amplification:

10X Amplification buffer, 10 ml

Mixture of four dNTPs (1.25 mM each), 17 ml

Forward primer (100 to 110 pmol) in dd.H2O, 4 ml

Reverse primer (100 to 110 pmol) in dd.H2O, 4 ml cDNA synthesized = 6 ml (0.1 to 1 mg)

Add dd.H2O to final volume of 100 ml.

For no cDNA control:

10X Amplification buffer, 10 ml

Mixture of four dNTPs (1.25 mM each), 17 ml

Forward primer (100 to 110 pmol) in dd.H2O, 4 ml

Reverse primer (100 to 110 pmol) in dd.H2O, 4 ml

Add dd.H2O to final volume of 100 ml.

b. Add 2.5 units of high fidelity of Taq DNA polymerase (5 units/ml) to each of the samples and mix well.

c. Overlay the mixture with 30 ml of light mineral oil (Sigma or equivalent) to prevent evaporation of the sample.

d. Carry out PCR amplification for 35 to 40 cycles in a PCR cycler programmed as follows:

Note: The condition for amplification should be exactly the same for every sample — actin cDNA as well as no cDNA.





First Subsequent Last

e. Starting after 10 cycles, carefully insert a pipette tip through the oil and remove 15 ml of reaction mixture from each of the samples and from controls every five cycles at the end of the appropriate cycle (at 70°C extension phase). Place tubes at 4°C until use.

Note: Sampling should be done in 2 min for all samples. There should be six samplings for each of the samples in 40 cycles. Oil should be avoided as much as possible.

4. Use the 15 ml cDNA from each of the samples and the controls to carry out dot blot hybridization using the appropriate 32P-labeled internal oligonucleotides or the target sequence (e.g., cDNA fragment) between the two primers used as a probe.

a. Denature DNA at 95°C for 10 to 15 min and immediately chill on ice. Spin down and add one volume of 20X SSC to the sample. An alternative is the alkaline denaturing method: add 0.2 volume of 2 M NaOH solution to the sample, incubate at room temperature for 15 min and add one volume of neutralization buffer containing 0.5 M Tris-HCl (pH 7.5) and 1.5 M NaCl. Incubate at room temperature for 15 min.

b. Cut a piece of nylon or nitrocellulose membrane filter and place the filter on top of a vacuum blot apparatus. Turn on the vacuum slightly to obtain an appropriate suction that holds the membrane filter onto the apparatus.

Tips: If the vacuum is too weak, diffusion of the loaded samples is usually a problem. However, the vacuum cannot be too strong; if the vacuum is too great, the efficiency of blotting decreases. Using an appropriate vacuum speed, a trace of each well will be visible on the membrane.

c. Spot samples (about 2 ml/loading without vacuum or 5 ml/loading under vacuum) onto the membrane filter that is prewetted with 10X SSC and air-dried. Partially dry the filter between each spot.

d. After spotting is complete, dry the filter under vacuum and place it onto a 3MM Whatman filter paper saturated with denaturing solution with the DNA side up. Incubate for 5 to 10 min.

e. Transfer the membrane filter, DNA side up, to a piece of 3MM Whatman filter paper presaturated with neutralizing solution for 2 to 5 min.

f. Air-dry the membrane filter at room temperature for 20 min.

g. Wrap the filter with SaranWrap and place DNA side down onto a transilluminator (312 nm wavelength is recommended) for 4 to 6 min for UV cross-linking.

h. Bake the filter in an oven at 80°C for 2 h under vacuum. Proceed to hybridization.

5. Carry out hybridization as described in Chapter 7.

6. Measure the signal intensities of dot spots with a densitometer. For the cDNA controls, no signals should be visible. For actin cDNA, the signal intensities should increase with amplified cycles, but no differences should be seen for any cell or tissue type or all developmental stages, or treated and untreated tissues. In contrast, if the expression of a specific gene is nonconstitutive but inducible with cell- or tissue-specific or chemical treatments, clear signal patterns of amplified cDNA dot blot hybridization can be seen. The signal for each sample should be stronger with amplified cycles (Figure 10.4).

7. If it is necessary to determine the size of PCR products, the cDNAs can be analyzed using 1.0 to 1.4% agarose gel electrophoresis and Southern blot hybridization using the target sequence as an internal probe (Figure 10.5).

Reagents Needed

First-Strand 5X Buffer

50 mM MgCl2

250 mM KCl

2.5 mM Spermidine

50 mM DTT

5 mM Each of dATP, dCTP, dGTP, dTTP

PCR Cycle mRNA Expression Actin mRNA as a control

Treated Untreated Treated

10 15

Time-course of treatment (hours) FIGURE 10.4 Dot blot hybridization showing expected semiquantitative PCR products.

mRNA Expression Actin mRNA as a control

--Treated Untreated Treated

10 15

20 25 30

24 48 72 24 48 72 24 48 72 Time-course of treatment (hours) FIGURE 10.5 Southern blot hybridization showing expected semiquantitative PCR products.

10X Amplification Buffer

500 mM KCl

15 mM MgCl2


1. Following electrophoresis, distribution of RNA species stained by EtBr is near the very bottom in some lanes instead of a long smear ranging from the top to the bottom of the lanes. This is due to obvious RNA degradation by RNase during RNA isolation, RNA storage or elec-trophoresis. Ensure that RNAs are handled without RNase contamination.

2. Two rRNA bands are not sharp, but instead are quite diffused. The voltage used for electrophoresis is too low and the gel is run for too long. To obtain the best results (sharp bands), a gel should run at an appropriate voltage.

3. After transfer is complete, obvious RNA staining remains in the agarose gel. RNA transfer is not efficient or complete. Try to set up the RNA transfer carefully and allow blotting to proceed for a longer time.

4. The blotted membrane shows some trace of bubbles. Obviously, this problem is due to bubbles generated between the gel and the membrane. Carefully follow instructions when assembling the blotting apparatus.

5. No signal is detected at all. This is the worst situation in northern blot hybridization. RNAs may not be effectively denatured or dsDNA probes mRNA Expression Actin mRNA as a control

--Treated Untreated Treated

may not be denatured prior to hybridization. When this happens, it is not surprising to see zero hybridization signals. Keep in mind that ssDNA probes and denatured RNAs are crucial for hybridization.

6. Hybridized signals are quite weak on the film or the filter. The activities of DNA probes may be low or hybridization efficiency is not good. To overcome this, allow hybridization to proceed for a longer time or increased exposure time to the x-ray film or develop longer on the filter.

7. Detected signals are not sharp bands; rather, long smears appear in each lane. This problem is most likely caused by nonspecific binding. Try to carry out hybridization and washing under high-stringency conditions.

8. Black background occurs on the x-ray film as revealed by chemilu-minescent detection. Multiple factors may be responsible for such a common problem. The membrane filter may have dried out during hybridization or during the washing process. Excess detection solution may not have been wiped out prior to exposure. Exposure time may be too long. The solution to this problem is to make sure that the filter is kept wet and that excess detection reagents are completely removed by wiping. Try to reduce exposure time for x-ray film.

9. A highly purple/blue background occurs on the filter as shown by colorimetric detection. Excess detection reagents may be used or the color is allowed to develop too long. Try using an appropriate amount of detection reagents and pay close attention to the color development. Once major bands become visible, stop development immediately by rinsing the filter with distilled water several times.

10. Unexpected bands show up on the x-ray film or the membrane filter. This problem is most likely caused by nonspecific binding between probes and the DNA species. To solve or prevent such a problem, one may increase blocking time for the blotted membrane and elevate stringency conditions for hybridization and washing processes.


1. Wu, W., Electrophoresis, blotting, and hybridization, in Handbook of Molecular and Cellular Methods in Biology and Medicine, Kaufman, P.B., Wu, W., Kim, D., and Cseke, L., pp. 87-122, CRC Press, Boca Raton, FL, 1995.

2. Wu, W. and Welsh, M.J., Expression of the 25-kDa heat-shock protein (HSP27) correlates with resistance to the toxicity of cadmium chloride, mercuric chloride, cis-platinum(II)-diammine dichloride, or sodium arsenite in mouse embryonic stem cells transfected with sense or antisense HSP27 cDNA, Toxicol. Appl. Pharmacol., 141, 330, 1996.

3. Welsh, M.J., Wu, W., Parvinen, M., and Gilmont, R.R., Variation in expression of HSP27 messenger ribonucleic acid during the cycle of the seminiferous epithelium and co-localization of HSP27 and microfilaments in Sertoli cells of the rat, Biol. Reprod, 55, 141-151, 1996.

4. Wu, (W.)L., Song, I., Karuppiah, R., and Kaufman, P.B., Kinetic induction of oat shoot pulvinus invertase mRNA by gravistimulation and partial cDNA cloning by the polymerase chain reaction, Plant Mol. Biol., 21(6): 1175-1179, 1993.

5. Wu, (W.)L., Mitchell, J.P., Cohn, N.S., and Kaufman, P.B., Gibberellin (GA3) enhances cell wall invertase activity and mRNA levels in elongating dwarf pea (Pisum sativum) shoots, Int. J. Plant Sci., 154(2): 278-288, 1993.

6. Wu, L., Song, I., Kim, D., and Kaufman, P.B., Molecular basis of the increase in invertase activity elicited by gravistimulation of oat-shoot pulvini, J. Plant Physiol., 142: 179-183, 1993.

7. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, John Wiley & Sons, New York, 1995.

8. Krumlauf, R., Northern blot analysis of gene expression, in Gene Transfer and Expression Protocols, Murray, E.J., Ed., The Human Press Inc., Clifton, NJ, 1991.

9. Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989.

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