Genetics and Genomics Molecular Biology

scientificprotocols authored over 3 years ago

Authors: Xin Wen, Wen Wei Zeng & Jack A. Yanovski

Introduction

TSS (Transcription starting site) determination is key to the study of transcriptional regulation, and “New 5´ RACE” (rapid amplification of cDNA ends) is a challenge method for determining multiple transcription starting sites . Since Melanocortin 3 receptor (MC3R) plays an important role in the regulation of body obesity, protocol for obtaining 5´UTR Transcription starting site of mouse Melanocortin 3 Receptor is presented.

Introduction

The 5´ends of transcripts give important information about transcription initiation sites ; RACE (rapid amplification of cDNA ends) PCR is useful for quickly obtaining full length cDNA from mRNA for which only part of the sequence is known. And “new 5´RACE” is changed by adding an anchor ligation to the 5´end of the mRNA before reverse transcription, thus, the anchor sequence becomes incorporated into the first-strand cDNA if and only if the reverse transcription proceeds through the entire length of the mRNA of interested . On the other hand, promoter of Melanocortin receptors are among those promoters of G-protein coupled receptors, which usually characterized as TATA less, GC rich. Melanocortin 3 receptors and Melanocortin 4 receptors are involved in the regulation of energy homeostasis. Therefore, it should be significant to determine their transcription starting site.

Since the intact mRNA has a 7-methyl guanosine CAP (figure1) on its 5´end. The 5´RACE could target its intact mRNA with improved specificity, through dephosphorylation with Calf Intestinal Alkaline Phosphatase (CIAP) to dephosphorylate degraded uncapped mRNA (figure1), then Tobacco Acid Pyrophophatase (TAP) treatment to remove cap from full length mRNA (Figure 2), and 5´RACE adaptor ligation to decapped mRNA. Due to the advantage of the adaptor, the “new 5´RACE” could produced the first strand cDNA by reverse transcription from the full length mRNA of targeted. With the nested PCR, it is like that the length of the primer become longer (the length of outer Plus that of inner primer), so that the contamination could be reduced and a higher specificity to the target sequence could be ensured. With TOPO Clone (Ligation) and Transformation, they are sent for sequencing. The transcription starting site could be determined from the statistics of the sequencing results.

There are 5’RACE for TSS determination for promoter of estrogen receptor , of human PPARg gene , etc. But there is no detail report of 5’RACE determination of TSS on Melanocortin 3 Receptor. Here a detail protocol based on our first hand experiments is disclosed. (Figure 2)

Discussion

Primer Design

Alter the stringency while designing primers so that all of the resulted three factors (hairpin, dimmers and false priming) meet the standards for primer design guidelines. (As it shown in tableA2-primer analysis result by primer premier, the experience should be -8.0kcals/mol, though we have -10.9kcal/mol for A-O-318 in the false primer, and -16.5kcal/mol for S-In-28, it still works).

In the primer design for the gene specific inner control primer 3, we designed two inner control primers (3a and 3b) instead of one, this way, we could get better primer match and easy way for primer design for the case of mMC3r. As a result, it shows (Table A2) good in analysis result as well as experiment. In addition, since the commercial available RACE kit has its limitations3, commercial systems are often geared toward the construction of universal pools of full-length cDNAs. So the use of GSP-RT primer, gives more potency for the reverse transcription steps starting more specifically. (Figure E, l and A1’c is from GSP-RT 496)

Reverse Transcription

Other than GSP-RT-601(figure E, A1’d), the GSP-RT-496 (figure E, A1’c) starts closer to the 5’end of transcripts and result a higher specific cDNA.

Besides, the 5’UTR of mMC3R gene is characterized as a TATA less and GC rich. (as high as 58.5% near the CpG island, detail data not shown here), Therefore, in order to get a fully open, instead of using M-MLV Reverse transcriptase in the kit, we used the SuperScript III Reverse transcriptase, (it is an engineered version of M-MLVR, reduced RNAse H activity and increased thermal stability, can be used to synthesize first-strand cDNA at temperature up to 55°C, providing increased specificity, higher yield of cDNA and more full length product than other reverse transcriptase.)

Outer and Nested PCR

In each PCR, the Touchdown PCR (Table Db, Table Eb) is used. It could avoid low Tm priming during the earlier cycles. Touchdown PCR increase specificity and reduced background amplification. By starting at a high annealing temperature, only gene-specific cDNA is amplified, allowing the target product to accumulate. Decreasing the annealing temperature through the remaining PCR cycles permits efficient amplification of tagged, gene-specific template. Since the empirical screening by determination of annealing temperature is time consuming, especially for the case of small amount presenting target template. As a result, Touchdown PCR is not only a method for determining the optimum cycling conditions for a specific PCR but also as a potential one-step method for approaching optimal amplification, specially, when the degree of identity between the primer and template is unclear.

Sequence and statistics

The consensus transcription start site -368 upstream of ATG should be identified.

Reagents

  1. Mouse Brain, hypothalamus Total RNA is from Clontech
  2. RLM-RACE kit from Ambion
  3. Transcription super III from Invitrogen
  4. Platinum PCR SuperMix High Fidelity is from Invitrogen
  5. SuperScriptIII RT is from Invitrogen
  6. TOPO Cloning kit is from Invitrogen
  7. One shot Efficiency DH5a-T1 Competent Cells for transformation is from Invitrogen
  8. EcoRI is from New England Biolabs
  9. DNA extractions kits are from Qiagen

Procedure

  1. Check the quality of the RNA and design primers as decribed in Protocol step A
  2. Perform RNA processing (B), Reverse Transcription (C) and Outer 5’RLM-RACE PCR (D) as described in Protocol step B, step C and step D.
  3. Perform Nested PCR(2+4) as described in Protocol step E
  4. Perfom TOPO TA Cloning as described in Protocol step F

Timing

This way, an ideal result could be offered by this protocol within a week (if every step is smooth).

Anticipated Results

Multiple transcriptional start sites may be identified in addition to the major site at position -368 from the initiating methionine that corresponds to the predicted transcription start site according to GenBank.

In a word, on the base of general steps of new 5’RACE, we use more RNA than what is suggested in the TAP treatment, use GS-RT Primer, SuperScriptIII and a higher temperature in the reverse transcription reaction. Two gene specific inner control sense primers are designed and used instead of one, with Platinum PCR Supermix, the reagent taking and mixing time for either outer PCR and nested PCR was reduced. Further more, with the touchdown PCR, for each of the outer and nested PCR processing, the time for screening and optimum the PCR reaction condition was also saved.

References

  1. Scotto-Lavino E, Du G, Frohman MA. Amplification of 5’ end cDNA amplification with “new RACE”. Nat.Protoc. 2006, 1(6):3056-61.
  2. Takeuchi S, Takahashi S. A possible involvement of melancortin 3 receptor in the regulation of adrenal gland function in the chicken. Biochem Biophys Acta. 1999 Jan 11; 1448(3); 512-8.
  3. Scotto-Lavino E, Du G, Frohman MA. Amplification of 5´ end cDNA amplification using classic RACE. Nat.Protoc. 2006, 1(6):2555-62.
  4. Y.Zhang, Frohman MA. Using rapid amplification of cDNA ends (RACE) to obtain full-length cDNAs. Methods in Molecular Biology. 1997, Springer. 69:61-87.
  5. Moro O, Ideta R, Ifuku O. Characterization of the promoter region of the human melanocortin-1 receptor gene. Biochem Biophys Res Commun. 1999 Aug 27; 262(2); 452-60.
  6. Rouzaud F, Hearing VJ. Regulatory elements of the melanocortin 1 receptor. Peptides. 2005 Oct; 26(10); 1858-70.
  7. MacNeil DJ, Van der Ploeg LH et al. The role of melanocortins in body weight regulation: opportunities for the treatment of obesity. Eur J Pharmacol. 2002 Aug 16; 450(1); 93-109.
  8. Ambion, Inc. FistChoise RLM-RACE kit, www.ambion.com/techlib/prot/fm_1700.pdf.
  9. Dumont LM, Wu CS, Aschkenasi CJ, Elmquist JK, Lowell BB, Mountjoy KG. Mouse melanocortin-4 receptor gene 5´-flanking region imparts cell specific expression in vitro. Mol Cell Endocrinol. 2001 Nov 26; 184(1-2): 173-85.
  10. Grandien K. Determination of transcription start sites in the human estrogen receptor gene and identification of a novel, tissue-specific, estrogen receptor-mRNA isoform. Mol Cell Endocrinol. 1996 Feb 5; 116(2); 207-12.
  11. Fajas L, Auwerx J. et al. The organization, promoter analysis, and expression of the human PPARgamma gene. J Biol Chem. 1997 Jul 25; 272(30); 18779-89.
  12. Invitrogen Topo Cloning Kit, http://tools.invitrogen.com/content/sfs/manuals/topota_man.pdf
  13. Invitrogen Gene Racer kit, http://tools.invitrogen.com/content/sfs/manuals/generacer_man.pdf
  14. Roux KH. Optimization and troubleshooting in PCR. PCR Methods Appl. 1995 Apr; 4(5); S185-94.
  15. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991 Jul; 19(14); 4008.

Acknowledgements

Thanks advice from Dr. Greti Aguilera, Dr. Yasuhiro Katagiri, tech support from Dr. Hong Lu, Ms.Ayelet Spitzer, Dr.Ying Liu, and thanks also to Dr. Susan Yanovski.

Figures

Protocol Step A:

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Protocol Step B:

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Protocol Step C:

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Protocol Step D:

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Protocol Step E:

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Protocol Step F:

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Figure 1:

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Figure 2:

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Figure D: Figure for Step D

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Figure E: Figure for step E

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Author information

Xin Wen, Wen Wei Zeng & Jack A. Yanovski, Unit on Growth and Obesity, PDEGEN, NICHD

Source: Protocol Exchange (2010) doi:10.1038/nprot.2010.28. Originally published online 16 February 2010.

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