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«Mikhail V. Matz Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117871 Moscow, Russia; Present address: Whitney laboratory, University ...»

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Amplification of representative cDNA samples from microscopic amounts of invertebrate

tissue to search for new genes

Mikhail V. Matz

Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117871 Moscow, Russia;

Present address: Whitney laboratory, University of Florida, 9505 Ocean Shore Blvd, St Augustine, FL 32080-8610.

email: matz@whitney.ufl.edu


Recently, we cloned six new GFP-like fluorescent proteins from five species of Antozoa (1),

including one red-emitting variant, DsRed, which is now commercially available. This project did not require expeditions and collection of animals on reefs: in all cases the starting material was just several milligrams of tissue (for example, a tentacle tip of a sea anemone), collected from a specimen in a private aquarium. This truly non-invasive kind of study was possible due to the approach of total cDNA amplification, which we extensively apply to various tasks and biological models in our lab. In this chapter I will outline our several-year experience in this helpful technique.

The possibility to amplify total cDNA obtained from small amounts of biological material is not yet routinely considered, despite the fact that obtaining amounts of material suitable for direct processing by standard methods is often time-consuming, expensive and may be even impossible. Perhaps the most significant obstacle to the full appreciation of the technique is the widespread belief that PCR amplification severely distorts the original cDNA profile, so that some cDNA species dramatically rise in abundance while others diminish and may even become completely lost. However, we found that there are just a few simple rules which should be followed to ensure that the amplified sample is minimally distorted and fully representative, i.e. contains all types of messages originally present in RNA, even the least abundant ones.

This was demonstrated in our own experiments on differential display (Fig. 1), and elsewhere in application of amplified cDNA as a probe for gene profiling by array technology (2-5). According to our experience in gene hunting in various biological models, amplified cDNA can substitute for normal, nonamplified cDNA in virtually all tasks. Moreover, in PCR-based gene hunting techniques such as RACE (6, 7), subtraction (8) or differential display (9) the amplified cDNA usually outperforms the normal one, because all backgrounds are predictable, and can be easily kept under control.

1.1. Total RNA isolation We usually use the following procedure, rather than commercial kits, because this technique is suitable for virtually all animals. It is based on the well-known protocol of Chomczynski and Sacchi (10), with one difference: all the procedures are performed at neutral pH instead of acidic as it was originally suggested. Also, the step of RNA precipitation with lithium chloride (LiCl) is added, because it results in very stable RNA preparations and considerably improves the consecutive procedures of cDNA synthesis.

We have successfully applied the protocol to RNA isolation from representatives of 13 phyla of multicellular animals. As an alternative, a popular Trizol method (GIBCO/Life Technologies) may be used in many cases, although it may not perform well on some non-standard objects, such as jellyfish. Kits for RNA isolation that utilize columns (such as Qiagen's RNeasy kit) are generally not recommended for nonstandard samples.

The protocol is designed for rather large tissue samples (tissue volume 10-100 µL), which normally yield about 10-100 micrograms of total RNA. The protocol for really microscopic amounts of starting material (expected to yield about 1 µg RNA or less) is the same but does not include second phenol-chloroform extraction (step 4) and LiCl precipitation (step 6). Additionally, the final “pellet” should be dissolved in 5 µL instead of 40 µL of water and transferred directly to cDNA synthesis, omitting the agarose gel analysis.

1. 2. cDNA synthesis We provide two alternatives for preparing amplified total cDNA from the isolated RNA, method A and method B (Fig. 2). Both methods provide a possibility to amplify a cDNA fraction corresponding to messenger (polyA[+]), RNA, starting from total RNA. The fraction of ribosomal RNA in the amplified sample, as it was determined in EST sequencing project based on amplified cDNA, is 15-20 %, represented mostly by small subunit ribosomal RNA. This is the same figure that is normally obtained with standard methods of cDNA synthesis (11).

Method A (“classical”) is to synthesize a double-stranded cDNA by a conventional means (employing DNA polymerase I / RNAseH / DNA ligase enzyme cocktail for second-strand synthesis), then ligate adaptors and amplify the sample using adaptor-specific primers. The structure of the adaptors evokes a PCR-suppression effect (12) and provides a method for selective amplification of only those cDNA molecules that contain both adaptor sequence and T-primer sequence, that is, corresponding to the polyA(+) fraction of RNA. The principles behind this method are described (13). The obvious advantage of this method is its high efficiency. A representative cDNA sample (with representation of 107 and higher) can be prepared from as little as 20-30 nanograms of total RNA. However, the method is rather laborious.

Method B is implemented in the SMART cDNA synthesis kit available from Clontech. It utilizes one surprising feature of Moloney murine leukemia virus reverse transcriptase (MMLV RT), its ability to add a few non-template deoxynucleotides (mostly C) to the 3' end of a newly synthesized cDNA strand upon reaching the 5' end of the RNA template. Oligonucleotide containing oligo(rG) sequence on the 3' end, which is called “template-switch oligo” (TS-oligo), will base pair with the deoxycytidine stretch produced by MMLV RT when added to the RT reaction. Reverse transcriptase then switches templates and continues replicating using the TS-oligo as a template. Thus, the sequence complementary to the TS-oligo can be attached to the 3' terminus of the first strand of cDNA synthesized, and may serve as a universal 5' terminal site for primer annealing during total cDNA amplification (14). Recently an improvement to the original procedure was reported (15). Addition of MnCl2 to the reaction mixture after first-strand synthesis, followed by a short incubation, increases the efficiency of non-template C addition to the cDNA and thus results in higher overall yield following cDNA amplification.

Although method B is simpler and faster than method A, its somewhat reduced efficiency means that a cDNA sample of suitable representation (more than 106) requires a minimum of one microgram of total RNA. It should be noted that both techniques (as they are described here) provide material not only for total cDNA amplification, but also for RACE (Rapid Amplification of cDNA Ends), a procedure for obtaining unknown flanks of a fragment. This procedure is indispensable for cloning complete coding regions of proteins. Different RACE techniques are available for each of the methods of cDNA amplification described here (refs. 6 and 7 for methods A and B respectively), both based on a PCR suppression effect (12).

2. Materials

2.1. Materials for Total RNA Isolation (see Note 1)

1. Dispersion buffer ("buffer D"): 4M Guanidine thiocyanate, 30 mM disodium citrate, 30 mM βmercaptoethanol, pH 7.0-7.5 (see Note 2).

2. Buffer-saturated phenol, pH 7.0-8.0 (GIBCO/Life Technologies).

3. Chloroform-isoamyl alcohol mix (24:1).

4. 96% ethanol.

5. 80% ethanol.

6. 12 M lithium chloride.

7. Co-precipitant: SeeDNA reagent (Amersham) or glycogen.

8. Fresh milliQ water.

9. Agarose gel (1%) containing ethidium bromide.

2.2. Materials for cDNA synthesis 2.2.1. Method A using conventional second strand synthesis (see Note 3)

1. SuperScript II reverse transcriptase, 200 U/µL (Life Technologies) or 20× PowerScript reverse transcriptase (Clontech) with provided buffer..

2. 0.1M DTT.

3. dNTP mix, 10 mM each.

4. 5× Second strand buffer: 500 mM KCl, 50 mM Ammonium sulfate, 25 mM MgCl2, 0.75 mM β-NAD, 100 mM Tris-HCl (pH 7.5), 0.25 mg/ml BSA.

5. 20× Second-strand enzyme cocktail: 6 U/µL DNA polymerase I, 0.2 U/µL RNase H, 1.2 U/µL E.coli DNA ligase.

6. T4 DNA polymerase (1-3 U/µL).

7. T4 DNA ligase 2-4 U/µL with provided buffer (New England Biolabs or equivalent).

8. T/M buffer: 10 mM Tris-HCl pH 8.0, 1 mM MgCl2.

9. Buffer-saturated phenol, pH 7.0-8.0 (GIBCO/Life Technologies).

10. Chloroform-isoamyl alcohol mix (24:1).

11. Long-and-Accurate PCR enzyme mix (Advantage2 polymerase mix by Clontech, LA-PCR by Takara, Expand Taq by Boehringer or equivalent, see Note 4).

12. 10× PCR buffer: provided with the enzyme mix or, if KlenTaq-based homemade mix is used: 300 mM tricine-KOH (pH 9.1), 160 mM ammonium sulfate, 30 mM MgCl2, 0.2 mg/ml BSA.

13. Yeast tRNA, 10 µg/µL.

14. 3M sodium acetate (pH 5).

15. Fresh milliQ water.

16. Agarose gel (1%) containing ethidium bromide.

17. Oligonucleotides: see Box 1 and Note 5.

2.2.2 Method B using template-switching effect

1. SuperScript II reverse transcriptase, 200 U/µL (Life Technologies) or 20× PowerScript reverse transcriptase (Clontech) with provided buffer.

2. 20 mM MnCl2.

3. 0.1M DTT.

4. dNTP mix, 10 mM each.

5. Long-and-Accurate PCR enzyme mix with buffer (see Note 4).

6. 10× PCR buffer: provided with the enzyme mix or, if KlenTaq-based homemade mix is used: 300 mM tricine-KOH (pH 9.1), 160 mM ammonium sulfate, 30 mM MgCl2, 0.2 mg/ml BSA.

7. Agarose gel (1%) containing ethidium bromide.

8. Fresh milliQ water.

9. Oligonucleotides: see Box 1 and Note 5.

3. Methods3.1 Total RNA Isolation

1. Dissolve the tissue sample in buffer D (see Note 6).

2. Spin the sample at maxumum speed on table microcentrifuge for 5 minutes at room temperature to remove debris. Transfer the supernatant to a new tube.

3. Put the tube on ice, add equal volume of buffer-saturated phenol and mix. There will be no phase separation at this moment. Add 1/5 volume of chloroform-isoamyl alcohol (24:1) and vortex the sample. Two distinct phases will separate. Vortex three to four more times with about one-minute intervals between steps. Incubate the tube on ice between steps. Spin at maxumum speed on table microcentrifuge for 30 minutes at +4 ºC. Remove and save the upper, aqueous phase. Take care to avoid warming the tube with your fingers, or the interphase may become invisible.

4. Repeat step 3.

5. Add 1 µL of co-precipitant, and then add an equal volume of 96% ethanol and mix. Spin immediately at maxumum speed on table microcentrifuge at room temperature for 10 minutes. The precipitate may not form a pellet, being instead spread over the back wall of the tube and thus being almost invisible even with co-precipitant added. Wash the pellet once with 0.5 ml 80% ethanol. Dry the pellet briefly until no liquid is seen in the tube (do not over-dry).

6. Dissolve the pellet in 100 µL of fresh milliQ water. If the pellet cannot be dissolved completely, remove the debris by spinning the sample at maxumum speed on table microcentrifuge for 3 minutes at room temperature. Transfer the supernatant to a new tube, then add equal volume of 12 M LiCl and chill the solution at –20 ºC for 30 minutes. Spin at maxumum speed on table microcentrifuge for 15 minutes at room temperature. Wash the pellet once with 0.5 ml 80% ethanol, and dry as previously done. The precipitated RNA is usually invisible, since co-precipitant does not precipitate in LiCl.

7. Dissolve the pellet in 40 µL of fresh milliQ water.

8. Load 2 µl of the solution onto a standard (non-denaturing) 1% agarose gel to check the amount and integrity of the RNA. Add ethidium bromide (EtBr) to the gel to avoid the additional (potentially RNAse-prone) step of gel staining. Load a known amount of some DNA on a neighboring lane to use as standard for determining the RNA concentration. Intact RNA should exhibit sharp band(s) of ribosomal RNA (see Fig. 3A and Notes 7-10).

3.2. Methods for cDNA Synthesis 3.2.1. Method A (“classical”) Method A first strand cDNA synthesis

1. To 5 µL RNA solution in water (0.03-3 µg of total RNA), add 1 µL of 10 µM primer TRsa and cover with mineral oil. Incubate at 65 ºC for 3 minutes, and then put the tube on ice.

2. Add 2 µL 5× first-strand buffer (provided with reverse transcriptase), 1 µL of 0.1M DTT, 1 µL of reverse transcriptese, 0.5 µL of dNTP mix (10 mM each) and incubate at 42 ºC for 1 hour, then put the tube on ice. Method A second strand cDNA synthesis

3. To the first-strand cDNA solution, add 49 µL of milliQ H2O, 1.6 µL of dNTP mix (10 mM each), 16 µL of 5× second strand reaction buffer, and 4 µL of 20× second-strand enzyme cocktail. (The total volume of the reaction mix is about 80 µL). Incubate at 16 ºC for 1.5 hours, and then put the tube on ice.

4. Add 1 µL T4 DNA polymerase, incubate 0.5 hours at 16 ºC to polish ends.

5. Stop the reaction by heating at 65 ºC for 5 minutes.

6. Take the reaction mix from under the oil, put in new tube and add 0.5 volume phenol then 0.5 volume chloroform-isoamyl alcohol (24:1). Vortex the solution and spin at maxumum speed on table microcentrifuge for 10 minutes. Transfer the upper, aqueous phase into new tube.

7. Add carrier (SeeDNA, Amersham, or glycogen) and precipitate DNA by adding 0.1 volume (8 µL) 3M sodium acetate (pH 5) and 2.5 volume (200 µL) 95% ethanol at room temperature. Spin immediately for 15 minutes at maxumum speed on table microcentrifuge at room temperature.

8. Wash the pellet with 80% ethanol; air-dry the pellet for about 5 minutes at room temp. Dissolve pellet in 6 µL H2O. Method A adaptor ligation

9. To the 6 µL of ds-cDNA, add 2 µL of adaptor (10 µM), 1 µL of 10× ligation buffer, 1 µL T4 DNA ligase and incubate overnight at 16 ºC.

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