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Rapid and Sensitive Analysis of mRNA Polyadenylation States by PCR

Fernando J. Sail,s 1 and Sidney Strickland

Department of Pharmacology, University Medical Center at Stony Brook, Stony Brook, New York 11794-8651

A rapid and sensitive technique is de- The length of the polyadenylate To facilitate our analysis of transla- scribed that measures the length of [poly(A)] tail on mRNAs has emerged as tional control of maternal mRNAs by cy- the poly(A) tall on a specific mRNA an important regulatory element. Previ- toplasmic polyadenylation, we have de- within subnanogram quantities of ous assays for polyadenylation have re- veloped a new assay that allows us to total cellular RNA [the Poly(A) test lied on Northern analysis of mRNAs fol- accurately determine the polyadenyla- (PAT)]. In a single-tube reaction, a lowing oligo(dT) hybridization and tion status of any mRNA in minute sam- poly(dT) primer is synthesized in situ RNase H cleavage (e.g., see Ref. 1). Com- ples. In this report we validate the on the poly(A) tall of mRNAs using parison of RNA samples in the presence poly(A) test (PAT) utilizing known mu- oligo(dT) and DNA Iigase. By modu- and absence of RNase H allows analysis rine maternal mRNAs and explore some lating the annealing temperature of differential polyadenylation states. of its potential applications. The PAT has and primer concentrations, a GC-rich This procedure has several drawbacks: also been used to establish Drosophila as adapter sequence is targeted to the (1) Because the RNA is detected by a model system for translational control S' end of the poly(dT) primer. This Northern analysis and several controls by polyadenylation. (4) Iigated poly(dT)-anchor is then used are required for each mRNA, a relatively The PAT provides many advantages to prime reverse transcription of large initial sample size is needed; (2) over previous poly(A) tail analyses: (1) the mRNA, yielding a library of PAT precise quantitation of poly(A) tail Speed--PAT can be performed in 1 day, cDNAs. The length of a poly(A) tail is length is difficult because of the limits of including RNA isolation and analysis of determined by PCR amplification us- conventional agarose gel electrophoresis PCR products; (2) ease--a sequential ing the oligo(dT)-anchor primer and used in Northern analysis; (3) extended one-tube reaction allows generation of a a message-spedfic primer. Comparl- adenosine residue stretches within the PAT cDNA library that can be used to an- son of PCR products from different RNA could complicate the analysis of alyze multiple RNAs; (3) sensitivity-- PAT samples allows quantitative determi- polyadenylation because they too can can be used for single-cell analysis; and naUon of changes in polyadenylation hybridize to oligo(dT) and be cleaved by (4) quantitation -- targeting an adapter of a given mRNA. This technique RNase H; and (4) multiple steps are sequence to the 5' end of the reverse overcomes many of the pitfalls asso- needed that potentially subject the RNA transcription primer in conjunction clated with conventional poly(A) tall to nonspecific degradation. with gel analysis of small PCR products length assessments and should prove Recently, other procedures have been allows accurate estimation of poly(A) tail useful in studying a variety of pro- described that improve on the RNase length. cesses relating to polyadenylation. H/oligo(dT) assay. Previously, we re- ported an assay [PER Poly(A) test] (z) that allows screening for changes in poly(A) MATERIALS AND METHODS tail length. This assay is fast and requires little initial material. However, the na- PAT cDNA Synthesis ture of the assay makes it difficult to ac- curately estimate poly(A) tail size. A sec- The acid-phenol method of mRNA puri- ond technique, RNA ligase-mediated fication was used (s) on 20-419 isolated rapid amplification of cDNA ends (RLM- primary oocytes and pooled aged sec- RACE), was used to determine the length ondary oocytes (6) with no additional car- of poly(A) tails in Tetrahymena thermo- rier RNA. However, any RNA isolation phila. (3) This technique has excellent procedure should be adequate, and we sensitivity and tail length quantitation; have also used the guanidine isothiocy- however, it involves multiple steps with anate method described in Huarte et intermediate RNA repurifications that al. (1) successfully. An aliquot [equivalent 1Present address: The Picower Institute for Medical can subject the RNA to degradation as well to 20-40 oocytes (8-16 ng of total Research, Manhasset, New York 11030. as a cumbersome single-stranded ligation. RNA] (7) of RNA in HzO was heat-dena-

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tured (65°C for 5 min) in a 7 Vd volume RNA in the presence of 20 ng of phosphory- lated oligo(dT) [p(dT)lz__ls] and placed 4z ° I 1)p(dT)12_18 ( TTTTT ~ directly at 42°C. Prewarmed mastermix [13 wl containing 4 wl of 5 x Superscript 2) T4 DNA ligase RNase H- reverse transcriptase (RT) (Ligation of oligo p(dT) subunits ) buffer (BRL); 2 Vd of 0.1 M DTT; 1 wl of 10 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA mM dNTPs; 1 ~1 of 10 mM ATP; 4 V.1 of TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT b, I-IzO; and I Vd of 10 U (Weiss)/wl T4 DNA ligase (U.S. Biochemical)] was added, / ~P~'- T'TTTT and the samples incubated at 42°C for 30 min. Subsequently, 1 wl of oligo(dT)-an- 12 ° ~ T/T-anchor (5 X) chor (200 ng/p,1, 5'-GCGAGCTCCGCG- GCCGCGT12 ) was added at 42°C and the (3' terrnmal targeting of anchor) reaction transferred to a 12°C water bath. AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA After a 2-hr incubation, the samples were TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT ~ transferred back to 42°C, 1 ~l (400 U/~l) TTT-anchor Superscript RNase H- RT was added, and / reverse transcription was performed for 42 ° ~ Reverse transcriptase 1 hr. cDNAs were diluted to 1 oocyte/pJ followed by a 30-min incubation at 70°C to inactivate the RT and ligase. A sche- (Reverse transcription of mRNA) matic representation of PAT cDNA syn- AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <-- TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT'r-an c h °r thesis is shown in Figure 1. An outlined protocol is provided in Table 1. PCR amplification

PCR Amplification mRNA specific pnmer TTT-anchor For PCR amplification, 1 Vd of PAT --c> cDNAs was added to a standard 50-~1 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-anchor PCR reaction spiked with 0.5 i~l of FIGURE 1 Schematic representation of the PCR-based PAT. Total RNA is prepared, and phospho- [3zp]dATP and containing 25 pmoles rylated oligonucleotide p(dT)lz_la is allowed to saturate the poly(A) tails of the mRNAs. The each of an mRNA-specific primer (see p(dT)lz_la is then ligated together at 42°C. This process generates a nearly full-length comple- legends) and the oligo(dT)-anchor [am- mentary copy of the poly(A) tail, leaving a short 3' overhang of oligo(A) as a result of the plification conditions: 93°C for 5 min; 30 instability of a short A/dT hybrid at 42°C. An oligo(dT)-anchor is then added at a fivefold molar cycles at 93°C for 30 sec; 62°C for 1 min; excess with respect to p(dT)12_1a, and the temperature is lowered to favor the hybridization and 72°C for 1 min; with a final extension of ligation of the oligo(dT)-anchor to the extreme 3' end of the poly(A) tail. After ligation, the 7 min at 72°C]. After amplification, PCR temperature is raised and the RNAs are reverse transcribed, using the ligated poly(dT)-anchor stretch as the template primer. The length of the poly(A) tail of a specific mRNA can be estimated products were phenol-extracted and eth- by the size of the PCR products after amplifying the cDNA using the oligo(dT)-anchor and a 5' anol-precipitated with 2.5 M ammonium primer specific for the mRNA of interest. acetate to remove unincorporated label. Samples were analyzed by autoradiogra- phy following electrophoresis on non- denaturing 5% polyacrylamide gels. RESULTS To ensure that the assay was accu- Screening for changes in poly(A) tail size rately measuring poly(A) tail length, the can also be performed easily without the PAT Analysis of mRNAs Known to PAT was applied to actin mRNA, an addition of radiolabeled nucleotide or Modulate Poly(A) Tail Length mRNA known to be deadenylated during the subsequent precipitations by ethid- the same time course that t-PA is polya- ium-bromide staining after agarose gel The assay was tested by examining the denylated (Fig. 3). (8) Actin mRNA con- electrophoresis (see Figs. 3 and 4, be- well-characterized dormant, maternal tained a poly(A) tail of -160 nucleotides low). To confirm the specificity of the mRNA encoding mouse tissue-type plas- in fully grown GV-containing oocytes amplification and that the size increase minogen activator (t-PA) (Fig. 2). t-PA and was essentially deadenylated by 6 hr of the PCR fragments was attributable to mRNA contains a poly(A) tail of -35 nu- after GV breakdown (GVBD) as deter- elongation at the 3' end of the mRNA, cleotides in fully grown germinal vesical mined by the PAT. These values are in the samples were digested with a restric- (GV)-containing oocytes. During oocyte agreement with those obtained previ- tion endonuclease that cleaves at a maturation, the poly(A) tail increases to ously by Northern analysis/s) known location from the 5' end of the -365 nucleotides. Estimates derived by amplified region (data not shown, see the PAT assay of both the extent and the PAT Analysis of Differential Ref. 2). This digestion should show a timing of polyadenylation are similar to Polyadenylation constant 5' fragment present in all time those determined previously from points and a heterogeneous 3' end. Northern analysis. °) In Xenopus, maternal mRNAs display dif-

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TABLE 1 PAT Protocol adenosine residue stretch (<~10) should remain because of the instability of a 1. Isolate total RNA. short (A-dT) annealed complex at 42°C. 2. Add 20 ng of oligo p(dT)lz_18 to the RNA to a final volume of 7 I~1. Heat-denature at 65°C At this point, an excess of unphosphory- for 5 min. Transfer samples immediately to 42°C without an ice-quenching step. lated oligo(dT)-anchor (5- to 10-fold 3. Add 13 ~1 of the following mixture (prewarmed to 42°C): 4 ~1 of S x Superscript H- RT buffer with respect to p(dT)lz__18) is added to 2 txl of 0.1 M DTT the reaction and the temperature is low- 1 I~1 of 10 mM each dNTP ered to 12°C. The lower temperature al- 1 ~1 of 10 mM ATP lows annealing of the oligo(dT) to the 4 ~1 of dHzO remaining short poly(A) stretch while 1 I~1 of 10 U/~I T4 DNA ligase the anchored primer excess favors the li- gation of the oligo(dT)-anchor versus 13 ~1 the p(dT)~z_18. After allowing sufficient Incubate for 30 min at 42°C. time to ligate the oligo(dT)-anchor, the 4. After step 3, while at 42°C, add 1 ~1 of 200 ng/l~l oligo(dT)-anchor. temperature is raised and reverse tran- 5. Vortex, microfuge, and incubate at 12°C for 2 hr. scription is performed using the nascent 6. Transfer the samples to 42°C for 2 min. poly(dT)-anchor as the primer. At the 7. Add 1 ~1 of Superscript H- RT. Incubate at 42°C for 1 hr. end of the reaction, the ligase and RT are 8. Dilute cDNAs to desired concentration. Heat to 70°C for 30 rain to inactivate ligase. heat-inactivated to prevent further mod- 9. PCR ifications during subsequent manipula- tions of the PAT cDNAs. For our experi- ments, samples are diluted to one oocyte or embryo per microliter. One (equiva- ferences in both the timing and extent of the initial ligation reaction. The oli- lent to 350-430 pg initial total RNA) (7) to polyadenylation. <9'~°) Two novel mouse go(dT) must be phosphorylated for the several microliters of sample can then be maternal mRNAs, cloned on the basis of ligation reaction. We use p(dT)~z_18 be- directly PCR amplified using an mRNA- altered polyadenylation (OM1 and cause we feel a heterogeneous mixture specific primer and the oligo(dT)-an- OM2), ~z) were subjected to the PAT. PAT will yield cleaner results by allowing chor primer. analysis demonstrates that these mouse greater ligation of the variable sized gaps mRNAs are polyadenylated to different between multiple elongating poly(dT) extents over a similar time course during chains (see below). The amount of PCR and Gel Analysis meiotic maturation (Fig. 4). Both con- p(dT)lz_~8 in the reaction should be in Theoretically, the message-specific tained a poly(A) tail of -60 nucleotides excess, as too little oligo(dT) or too primer can be directed anywhere along in fully grown GV-stage oocytes. During much RNA will prevent complete liga- the mRNA. Primer locations close to the meiotic maturation, OM 1 receives - 170 tion of oligonucleotide subunits and 3'end (within 400 nucleotides) are pref- adenosine residues, whereas OM2 re- possibly lead to misrepresentation of the erable because they provide the best PCR ceives -590. As in the case of t-PA and actual poly(A) tail lengths (see below). product size resolution. An internal actin, these data are in agreement with The amount of ligase used is in excess to primer can be used followed by restric- those derived from Northern analysis ensure significant activity after 30 min at tion digest to allow visualization of the [OMl~Z); OM2°~)]. Combined, these re- 42°C for ligation of the oligo(dT)-an- PCR products representing the 3' end of suits demonstrate that the PAT accu- chor, which is critical, as the anchor is the mRNA. °z~ We use the primer pro- rately determines the polyadenylation required for proper amplification of the gram contained in the Genetics Com- state of mRNAs. 3' terminus and poly(A) tract. puter Group (GCG) software to identify Theoretically, the p(dT)~z__~8 can an- suitable sequences. We recommend neal anywhere on the poly(A) tail. At primers that range between 20 and 25 DISCUSSION 42°C, the subunit annealing should be nucleotides, 40%-60% GC, Tm -60°C weak and allow transient melting and and that do not have significant compli- PAT Theory sliding along the poly(A) tract. Ligation mentarity to the oligo(dT)-anchor. If We routinely perform RNA isolations will occur when two subunits abut each spurious amplification products are a from cells in the absence of carrier even other in the presence of ligase. Ligation problem, the annealing temperature in when dealing with minute samples (e.g., will increase the stability of the elongat- the PCR can be increased slightly 20 oocytes, 8 ng of total RNA). The pres- ing chain of subunits along the poly(A) (2-5°C). If modifying the PCR condi- ence of carrier should not affect the re- tract, limiting further sliding of the na- tions fails to give satisfactory results, a sults, although it is possible that carrier scent poly(dT) tract. ~ At the termini of primer at a different location can be excess will compete for oligo(dT) during the poly(A) tail, a small single-stranded tried.

1In the case of long poly(A) tails, it is likely that two or more poly(dT) chains will be initiated simultaneously on different regions of the poly(A) tail. Given that the oligo(dT) is 12-18 nucleotides in length, it is likely that the chains will leave a gap too small to incorporate an oligo(dT) subunit. When the temperature is lowered, the oligo(dT)- adapter may ligate with the 5' phosphates of each poly(dT) chain, allowing subsequent PCR amplification from what would appear upon electrophoresis as an internal position on the poly(A) tail. This would lead to a "laddering" effect, which we have observed (see Troubleshooting). If oligo(dT)-adapter ligation does not occur, the subsequent individual cDNA should not be amplifiable.

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Hours after GVBD sized fragment, which represents the 5' portion of the mRNA of interest, in all GV 1 6 11 16 28 24GV samples (data not shown; see Ref. 2). Poly (A) Length Troubleshooting 365 Occasionally, a laddering of PCR prod- ucts between the expected minimum size and the maximally polyadenylated species is seen and appears to be indica-

4.m ...... 165 (/) tive of unsaturating p(dT)lz__lS concen- trations or multiple oligo(dT) ligations

O laddering generally varies with each PAT ~L 35 cDNA synthesis. In our experience, addi- tional bands outside of the expected range and/or excessive smearing are gen- erally indicative of PCR problems. In these instances, the PCR annealing tem- perature, the number of cycles or, fi- nally, the specific primer selection should be altered to optimize results and not the PAT cDNA synthesis. If problems persist, the RNAs should be DNase E 28s treated to ensure that artifacts do not ,.c arise from genomic or plasmid DNA con- L_ 0 tamination. We have successfully used Z the DNase treatment described in Vas- : 18S salli et al. (13) with the PAT assay. FIGURE 2 The PAT accurately determines poly(A) tail length. PAT was applied to the mRNA for mouse t-PA. (GV) GV-stage oocytes isolated at various times during oocyte maturation [hours in culture after GV breakdown (GVBD)]; (24GV) aged control GV-stage oocytes (prevented from undergoing maturation isolated after 24 hr in culture); these aged GV oocytes do not produce OM1 OM2 t-PA and do not polyadenylate the mRNA.(z) One oocyte equivalent of PAT cDNAs was amplified per time point. The autoradiograph represents an overnight exposure of 25% of the PCR products O I Oil - O I O.. (GV, 1 hr, 24 hr GV) or 50% of the products (6, 11, 16 hr). (t-PA primer) 5'-ACTCTATAGATGGT- TGGGAG. The minimum expected size of the amplified products is 185 bp [155-bp t-PA 3' end + 30 bp of oligo(dT)-anchor]. The estimates of polyadenylation derived from the PAT are in Poly (A) good agreement with those derived from Northern analysis of t-PA mRNA isolated from mouse Length ...... 650 oocytes over a similar time course (Bottom, taken from Ref. 1). Moreover, the PAT reflects the 230 levels of the mRNA over the time course presented when compared with Northern analysis. Estimates of size are derived from molecular weight standards.

GVBD (hrs) The PCR-amplified products can be FIGURE 4 PAT analysis of differential polya- analyzed by either denaturing or nonde- O~1 6 11 1628 denylation. PAT PCR products were stained Poly (A) naturing polyacrylamide gel or agarose with ethidium bromide after agarose gel elec- Length gel electrophoresis. We have found that trophoresis. Shown are two murine maternal 160 the use of a radiolabeled nucleotide in ...... mRNAs known to be polyadenylated to differ- 0 -' the PCR allows the most accurate deter- ent extents during oocyte meiotic matura- mination of tail length; interpretable re- tion. One oocyte equivalent was PCR ampli- FIGURE 3 PAT accurately determines deade- sults can also be obtained by ethidium fied per sample with a specific primer nylation of actin mRNA. PAT PCR products bromide staining of agarose gels (cf. Fig. [(OM1) 5'-ACCAGAAGTGCTAACCGGAATGT- were stained with ethidium bromide after 2 with Figs. 3 and 4). Additionally, we GA, (OM2) 5'-CATGTCCAGCTTTTAAGTGG]. agarose gel ele~-trophoresis. The PAT cDNAs find that with radiolabeled PCR, nonde- The minimum expected size of OMl-ampli- were the sam ones used in Fig. 2, and one fled products is 124 bp [90 bp of OM1 naturing polyacrylamide gels yield more oocyte equivalent was PCR amplified with an 3'end+30-bp oligo(dT)-anchor]. The mini- aesthetic results, whereas denaturing actin-specific primer (5'-AAGAGGAGGATG- mum expected size of OM2-amplified prod- GTCGCGTCCATGC). The minimum expected gels are more reliable. Finally, to con- ucts is 89 bp [59-bp OM1 3' end+ 30-bp oli- size of the amplified products is 323 bp [293- firm specific amplification the PCR prod- go(dT)-anchor]. (Oi) GV-stage oocytes; (O,) bp actin 3' end+ 30 bp of oligo(dT)-anchor]. ucts should be digested with a restriction oocytes that have completed meiotic matura- (Oi) GV-stage oocytes. endonuclease that will yield a constant tion, -16-20 hr after GVBD.

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CONCLUSIONS and S. Strickland. 1992. Isolation of novel 15. Zingg, H.H., D.L. Lefebvre, and G. A1- murine maternal mRNAs regulated by cy- mazan. 1988. Regulation of poly(A) tail The PAT has several advantages for the toplasmic polyadenylation. Genes & Dev. size of vasopressin mRNA. J. Biol. Chem. analysis of polyadenylation. (1) By using 6: 1201-1212. 263: 11041-11043. the PCR, the sensitivity of the assay is 3. Liu, X. and M.A. Gorovsky. 1993. Map- 16. Zingg, H.H. and D.L. Lefebvre. 1989. Ox- increased such that the analysis can be ping the 5' and 3' ends of Tetrahymena ytocin mRNA: Increase of polyadenylate performed on minute samples (fractions thermophila mRNAs using RNA ligase me- tail size during pregnancy and lactation. of a single oocyte or embryo). (2) The diated amplification of cDNA ends (RLM- Mol. Cell. EndocrinoL 65: 59-62. assay can be performed on total cellular RACE). Nucleic Acids Res. 21" 4954-4960. 17. Bhat, G.J., A.E. Souza, J.E. Feagin, and K. RNA. (3) The ligation and reverse tran- 4. Sall6s, F.J., M.E. Lieberfarb, C. Wreden, Stuart. 1992. Transcript-specific develop- J.P. Gergen, and S. Strickland. 1994. Co- mental regulation of polyadenylation in scription steps can be performed on a ordinate initiation of Drosophila develop- Trypanosoma brucei mitochondria. Mol. larger number of specimens and stored. ment by regulated polyadenylation of Biochem. Parasitol. 52: 231-240. When a specific mRNA is to be analyzed, maternal mRNAs. Science 266:1996- 18. Sch/ifer, M., R. Kuhn, F. Bosse, and U. a primer for that RNA is synthesized and 1999. Sch/ifer. 1990. A conserved element in the used in the PCR with the stored cDNAs. 5. Chomczynski, P. and N. Sacchi. 1987. Sin- leader mediates post-meiotic translation (4) The results can typically be visualized gle-step method of RNA isolation by acid as well as cytoplasmic polyadenylation of with ethidium bromide-stained 2% aga- guanidinium thiocyanate-phenol-chloro- a Drosophila spermatocyte mRNA. EMBO rose gel electrophoresis. For the analysis form extraction. Anal. Biochem. 162: 156- J. 9: 4519-4525. of rare mRNAs or if a more precise deter- 159. mination of the extent of polyadenyla- 6. Sall6s, F.J., W.G. Richards, J. Huarte, J.-D. Vassalli, and S. Strickland. 1993. Microin- tion is desired, the PCR reaction can be Received January 26, 199& accepted in jecting antisense sequences into oocytes. carried out in the presence of a radiola- revised form March 6, 1995. Methods Enzymol. 225: 351-361. beled nucleotide and separated on poly- 7. Bachvarova, R., V. De Leon, A. Johnson, acrylamide gels. (5) Given all the re- G. Kaplan, and B. V. Paynton. 1985. quired reagents, the assay can be done in Changes in total RNA, polyadenylated 1 day. RNA, and actin mRNA during meiotic Given the ease and sensitivity of this maturation of mouse oocytes. Dev. Biol. assay, it should prove useful for the anal- 108: 325-331. ysis of any mRNA in any tissue including 8. Paynton, B.V., R. Rempel, and R. Bach- those already shown to undergo changes varova. 1988. Changes in state of adeny- in polyadenylation [e.g. vasopres- lation and time course of degradation of maternal mRNAs during oocyte matura- sin (14As) and oxytocin (16) in mice; Cyb, tion and early embryonic development in COI, COII, and CR1 mRNAs in Trypano- the mouse. Dev. Biol. 129: 304-314. soma brucei, (17) MST87F in Droso- 9. Simon, R., J.P. Tassan, and J.D. Richter. phila(18)]. Additionally, the assay could 1992. Translational control by poly(A) be adapted for analyzing sequence or elongation during Xenopus development: structural requirements that regulate Differential repression and enhancement both timing and extent of polyadenyla- by a novel cytoplasmic polyadenylation tion using microinjected RNAs or with in element. Genes & Dev. 6: 2580-2591. vitro extracts as well as for the analysis of 10. Sheets, M.D., C.A. Fox, T. Hunt, G. Vande Woude, and M. Wickens. 1994. The 3'- related topics such as deadenylation of untranslated regions of c-mos and cyclin mRNAs. mRNAs stimulate translation by regulat- ing cytoplasmic polyadenylation. Genes & ACKNOWLEDGMENTS Dev. 8: 926-938. 11. West, M. and S. Strickland (unpubl.). We thank Michael Frohman, Marshal 12. Huarte, J., A. Stutz, M.L. O'Connell, P. Gu- Lieberfarb, Christopher Wreden, and bier, D. Belin, A.L. Darrow, S. Strickland, members of the Strickland laboratory for and J.-D. Vassalli. 1992. Transient transla- helpful discussions in developing the tional Silencing by Reversible mRNA PAT assay. This work was supported by a Deadenylation. Cell 69: 1021-1030. 13. Vassalli, J.-D., J. Huarte, D. Belin, P. Gu- National Institutes of Health grant bier, A. Vassalli, M.L. O'Connell, L.A. Par- (HD25922) to S.S. ton, R.J. Rickles, and S. Strickland. 1989. Regulated polyadenylation controls REFERENCES mRNA translation during meiotic matura- tion of mouse oocytes. Genes & Dev. 1. Huarte, J., D. Belin, A. Vassalli, S. Strick- 3: 2163-2171. land, and J.-D. Vassalli. 1987. Meiotic 14. Carranzana, E.J., K.B. Pasieka, andJ.A. Ma- maturation of mouse oocytes triggers the jzoub. 1988. The vasopressin mRNA translation and polyadenylation of dor- poly(A) tract is unusually long and in- mant tissue-type plasminogen activator creases during stimulation of vasopressin mRNA. Genes & Dev. 1: 1201-1211. gene expression in vivo. Mol. Cell Biol. 2. Sall6s, F.J., A.L. Darrow, M.L. O'Connell, 8: 2267-2274.

PCR Methods and Applications 321 Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

Rapid and sensitive analysis of mRNA polyadenylation states by PCR.

F J Sallés and S Strickland

Genome Res. 1995 4: 317-321

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