Nucleic Acid Research Group Study:

Transkript

1 Nucleic Acid Research Group Study: A Comparison of Different Priming Strategies for cdna Synthesis by Reverse Transcriptase as Measured by Real-Time RT-qPCR

2 NARG Survey Results for Types of Primers Used for RT Reactions Reverse Transcription Primer Used Random primers Oligo(dT) Gene-specific primer Random primers and oligo(dt) mixed Sample is provided # of respondents

3 Literature Suggested Long Randomers Were More Effective Than Short Randomers In Generating cdna Stangegaard, et. al., BioTechniques 40:649, 2006 Random Pentadecamer (15-mer) Assessed resultant cdna over a micorarray

4 Study Goal To evaluate priming strategies used in the reverse transcriptase (RT) reaction to make cdna for use in realtime qpcr Compare randomers, oligo(dt), anchored oligo(dt), and GSP Compare randomers of different lengths Compare combinations of randomers and Oligo (dt)

5 Reverse Transcriptase Method Primers (synthesized by Integrated DNA Technologies) Randomers: 6-mer, 9-mer, 12-mer, 15-mer, 18-mer, and 21-mer Oligo(dT) 20 and Anchored Oligo(dT) 20 All Randomer with oligo (dt) or anchored oligo (dt) combinations Gene Specific Primers: β-actin, β-glucuronidase, TATA Binding Protein No Primer and No Primer, No RT RNA FirstChoice Human Brain Reference RNA (Ambion) One of the same reference RNAs used as an External RNA Control (ERC) in the MAQC study (Nature Biotechnology: 24, , 2006) RT Assay SuperScript III cdna synthesis kit per manufacturer s (Invitrogen Corp) suggested protocol 100 ng total RNA/reaction Primer (final conc. for 20 μl rxn) Randomer: 4 μm Oligo (dt): 2.5 μm Anchored oligo (dt): 2.5 μm Gene specific: 100 nm Combos: 2 μm randomer + 2 μm oligo dt (or anchored oligo dt) cdna Synthesis Randomers and Randomer combos: 25 C, 10 min then 50 C, 50 min Oligo (dt), Anchored Oligo (dt), GSP: 50 C, 50 min Triplicate reactions

6 Bioanalyzer Analysis of the Study RNA

7 Quantitative PCR Methods qpcr Assay cdna: [Primer]: Probe: Set Up Method: Chemistry: Replication: 10 ng RNA equivalents from RT rxn Individual Lab specified Individual Lab specified Individual Lab specified Individual Lab specified Each RT rxn run in duplicate wells qpcr Results Collected Ct values ΔCt = Ct primer -Ct no primer

8 Study Design Human Brain Reference RNA used for Microarray Study 100 ng Total RNA per RT Reaction SuperScript III 25 RT priming conditions to test 3 genes: β-actin, β-glucouronidase, TATA Binding Protein Random primers (6) Random primers + Anchored (dt) (6) Random primers + Oligo (dt) (6) Oligo (dt) (1) Anchored Oligo (dt) (1) No primer +enzyme (1) No primer, no enzyme (1) Gene specific primers (3) RT reactions in triplicate Duplicate PCR reactions on each RT reaction Run with TaqMan Chemistries

9 Real-Time qpcr Assay Maps hβ-actin transcript hβ-actin bases 3 (NM_001101) Total length bases 71 bases 344 hβ-glucuronidase transcript 5 bases (NM_000181) hβ-gus 3 Total length bases 66 bases htata Binding Protein transcript 746 bases 5 (NM_003194) htbp 3 Total length bases 80 bases

10 Statistical Analysis The effect of each variable on Ct or ΔCt levels were assessed using a one-way analysis of variance (ANOVA) with the JMP v 5.01 Statistical Discovery Software (SAS Institute, Cary, NC). The green diamonds represent the mean and the standard error which is a pooled estimate of the variance. A Student s t-test was used to assess for significant difference levels (P < 0.05) between the groups contained within each variable.

11 ALL GENES (ASSAYS)

12 Effect of Primer on Ct (All Genes) Ct Ct 12mer 12mer/AnchOligo(dT) 12mer/Oligo(dT) 15mer 15mer/AnchOligo(dT) 15mer/Oligo(dT) 18mer 18mer/AnchOligo(dT) 18mer/Oligo(dT) 21mer 21mer/AnchOligo(dT) 21mer/Oligo(dT) 6mer 6mer/AnchOligo(dT) 6mer/Oligo(dT) 9mer 9mer/AnchOligo(dT) 9mer/Oligo(dT) AnchOligo(dT) GUS NoPrimer Oligo(dT) TBP b-actin Primer P <

13 Effect of Primer on Ct (All Genes) Primer Level* Mean Ct (N) NoPrimer A (101) TBP A B (33) 21mer B C (102) GUS B C (27) 15mer C (102) 18mer C (101) 15mer/Oligo(dT) C (100) 12mer C (102) 18mer/Oligo(dT) C (100) Oligo(dT) C (102) 15mer/AnchOligo(dT) C (102) AnchOligo(dT) C (99) 18mer/AnchOligo(dT) C (101) 21mer/AnchOligo(dT) C (102) 21mer/Oligo(dT) C (102) 12mer/Oligo(dT) C (102) 12mer/AnchOligo(dT) C (102) 6mer C (102) 6mer/AnchOligo(dT) C (101) 9mer/AnchOligo(dT) C (101) 9mer C (101) 6mer/Oligo(dT) C (102) 9mer/Oligo(dT) C (101) β-actin D (36) *Levels not connected by the same letter are significantly different (p<0.05)

14 delta Ct Ct 12mer 12mer/AnchOligo(dT) 12mer/Oligo(dT) 15mer 15mer/AnchOligo(dT) 15mer/Oligo(dT) 18mer 18mer/AnchOligo(dT) 18mer/Oligo(dT) 21mer 21mer/AnchOligo(dT) 21mer/Oligo(dT) 6mer 6mer/AnchOligo(dT) 6mer/Oligo(dT) 9mer 9mer/AnchOligo(dT) 9mer/Oligo(dT) AnchOligo(dT) GUS NoPrimer Oligo(dT) TBP b-actin Effect of Primer on ΔCt (All Genes) Primer P <

15 Effect of Primer on ΔCt (All Genes) Primer Level* Mean ΔCt (N) TBP A 4.95 (33) GUS A 4.56 (27) 9mer/Oligo(dT) B 3.45 (101) 9mer B 3.44 (101) 9mer/AnchOligo(dT) B 3.39 (101) 6mer/Oligo(dT) B C 3.37 (102) 6mer B C D 3.28 (102) 6mer/AnchOligo(dT) B C D 3.25 (101) 12mer/Oligo(dT) B C D 3.16 (102) 21mer/Oligo(dT) B C D 3.15 (102) 12mer/AnchOligo(dT) B C D 3.12 (102) 18mer/AnchOligo(dT) B C D E 3.10 (101) 18mer/Oligo(dT) B C D E 3.09 (100) 21mer/AnchOligo(dT) B C D E 3.08 (102) 15mer/AnchOligo(dT) C D E 2.98 (102) Oligo(dT) C D E 2.97 (102) 12mer D E F 2.92 (102) AnchOligo(dT) D E F 2.90 (99) 15mer/Oligo(dT) D E F 2.90 (100) 18mer E F G 2.71 (101) 15mer F G H 2.53 (102) β-actin G H 2.19 (36) 21mer H 2.17 (102) NoPrimer I 0.11 (101) *Levels not connected by the same letter are significantly different (p<0.05)

16 Effect of Primer Classification on Ct Primer Class Level* Mean (N) Rand A (610) O(dT) A B (102) Ct Ct ao(dt) A B (99) Rand/aO(dT) A B (608) Rand/O(dT) A B (607) GSP B (96) GSP GSP O(dT) O(dT) Rand Rand Rand/O(dT) Rand/aO(dT) ao(dt) ao(dt) Primer Primer Classification P = *Levels not connected by the same letter are significantly different (p<0.05).

17 Effect of Primer Classification on ΔCt Primer Class Level* Mean (N) GSP A 3.81 (96) Rand/O(dT) B 3.19 (607) delta delta Ct Ct GSP GSP O(dT) O(dT) Rand Rand Rand/O(dT) Rand/aO(dT) ao(dt) Primer Classification P < Rand/aO(dT) B 3.15 (608) O(dT) B C 2.97 (102) ao(dt) B C 2.90 (99) Rand C 2.83 (610) *Levels not connected by the same letter are significantly different (p<0.05).

18 Effect of Reverse Transcriptase Replicate on Ct Ct A B C RT Replicate P = RT Rep. Level* Mean (N) A A (737) B A (746) C A (740) *Levels not connected by the same letter are significantly different (p<0.05).

19 Effect of Reverse Transcriptase Replicate on ΔCt delta Ct A B C RT Replicate RT Replicate Level* Mean (N) C A 2.99 (740) A A 2.95 (740) B A 2.89 (746) *Levels not connected by the same letter are significantly different (p<0.05). P =

20 Effect of qpcr Replicate on Ct Ct qpcr Replicate qpcr Rep. Level* Mean (N) 1 A 26.4 (1,111) 2 A 26.3 (1,112) *Levels not connected by the same letter are significantly different (p<0.05). P =

21 Effect of qpcr Replicate on ΔCt delta Ct qpcr Replicate qpcr Replicate Level* Mean (N) 2 A 2.96 (1,111) 1 A 2.93 (1,112) *Levels not connected by the same letter are significantly different (p<0.05). P =

22 Effect of Set Up Method on Ct Ct Set Up Method Level* Mean (N) Manual A (1,307) 20 Manual Set up Method Robot P < Robot B (916) *Levels not connected by the same letter are significantly different (p<0.05).

23 Effect of Set Up Method on ΔCt delta Ct Manual Robot Set Up Method Level* Mean(N) Manual A 3.01 (1307) Robot B 2.86 (917) *Levels not connected by the same letter are significantly different (p<0.05). Set up Method P <

24 Effect of Lab on Ct Ct Lab A Lab B Lab C Lab D Lab E Lab F Lab G Site Lab Level* Mean (N) Lab A A (132) Lab D A (388) Lab E B (391) Lab G B (396) Lab F C (396) Lab B D (396) Lab C E (124) P < *Levels not connected by the same letter are significantly different (p<0.05).

25 For Labs that Performed All Assays- Effect of Lab on Ct Ct Lab Level* Mean (N) Lab D A (388) Lab E B (391) Lab G B (396) Lab F C (396) Lab B D (396) 20 Lab B Lab D Lab E Lab F Lab G Site (All Assays) *Levels not connected by the same letter are significantly different (p<0.05). P <

26 For Labs that Performed All Assays- Effect of Lab on ΔCt delta Ct Lab B Lab D Lab E Lab F Lab G Site (All Assays) Lab Level* Mean (N) Lab B A 4.40 (396) Lab E B 3.14 (391) Lab D B C 3.03 (388) Lab F C 2.88 (396) Lab G D 1.81 (396) *Levels not connected by the same letter are significantly different (p<0.05). P <

27 Effect of Gene (Assay) on Ct Ct Gene (Assay) Level* Mean (N) TBP A (787) GUS B (653) β-actin C (773) 20 GUS TBP b-actin Gene *Levels not connected by the same letter are significantly different (p<0.05). P <

28 Effect of Gene (Assay) on ΔCt delta Ct GUS TBP b-actin Gene Gene (Assay) Level* Mean (N) GUS A 3.58 (653) TBP B 3.20 (787) β-actin C 2.16 (773) *Levels not connected by the same letter are significantly different (p<0.05). P <

29 General Results There was a great variation in Ct and ΔCt between labs, though within lab variation between replicates was negligible The effect of RT biological replicates and qpcr replicates was not significant. The robotic set up gave significantly lower Ct and higher ΔCt values than manual set-up Assay Type (Gene) significantly affected Ct and ΔCt levels. The effects of primer or primer classification on Ct and ΔCt will be evaluated within each assay

30 β-actin hβ-actin hβ-actin transcript bases 3 Total length bases 71 bases 7 Stem Structures by m-fold

31 Amplification Plot for β-actin Randomers, Oligo(dT), aoligo(dt), Rand + O(dT), Rand + ao(dt) No Primer β-actin No Primer, No RT

32 Effect of Primer by Assay (β-actin) on Ct Ct mer 12mer/AnchOligo(dT) 12mer/Oligo(dT) 15mer 15mer/AnchOligo(dT) 15mer/Oligo(dT) 18mer 18mer/AnchOligo(dT) 18mer/Oligo(dT) 21mer 21mer/AnchOligo(dT) 21mer/Oligo(dT) 6mer 6mer/AnchOligo(dT) 6mer/Oligo(dT) 9mer 9mer/AnchOligo(dT) 9mer/Oligo(dT) AnchOligo(dT) Oligo(dT) b-actin Primer Type P =

33 Effect of Primer by Assay (β-actin) on Ct Primer Level* Mean 21mer A (36) 15mer/Oligo(dT) A B (35) 18mer A B (35) Oligo(dT) A B (36) AnchOligo(dT) A B (35) 12mer/AnchOligo(dT) A B C (36) 15mer A B C (36) β-actin A B C (36) 21mer/Oligo(dT) A B C (36) 18mer/Oligo(dT) A B C (34) 12mer/Oligo(dT) A B C (36) 18mer/AnchOligo(dT) A B C (35) 21mer/AnchOligo(dT) A B C (36) 15mer/AnchOligo(dT) A B C (36) 6mer/Oligo(dT) A B C (36) 12mer B C (36) 6mer/AnchOligo(dT) B C (36) 9mer/Oligo(dT) B C (35) 6mer B C (36) 9mer/AnchOligo(dT) B C (35) 9mer C (35) *Levels not connected by the same letter are significantly different (p<0.05)

34 Effect of Primer by Assay (β-actin) on Ct Ct mer 12mer/AnchOligo(dT) 12mer/Oligo(dT) 15mer 15mer/AnchOligo(dT) 15mer/Oligo(dT) 18mer 18mer/AnchOligo(dT) 18mer/Oligo(dT) 21mer 21mer/AnchOligo(dT) 21mer/Oligo(dT) 6mer 6mer/AnchOligo(dT) 6mer/Oligo(dT) 9mer 9mer/AnchOligo(dT) 9mer/Oligo(dT) AnchOligo(dT) NoPrimer Oligo(dT) b-actin Primer P <

35 Effect of Primer by Assay (β-actin) on Ct Level Mean (N) NoPrimer A (36) 21mer B (36) 15mer/Oligo(dT) B C (35) 18mer B C (35) Oligo(dT) B C (36) AnchOligo(dT) B C (35) 12mer/AnchOligo(dT) B C D (36) 15mer B C D (36) β-actin B C D (36) 21mer/Oligo(dT) B C D (36) 18mer/Oligo(dT) B C D (34) 12mer/Oligo(dT) B C D (36) 18mer/AnchOligo(dT) B C D (35) 21mer/AnchOligo(dT) B C D (36) 15mer/AnchOligo(dT) B C D (36) 6mer/Oligo(dT) B C D (36) 12mer C D (36) 6mer/AnchOligo(dT) C D (36) 9mer/Oligo(dT) C D (35) 6mer C D (36) 9mer/AnchOligo(dT) C D (35) 9mer D (35) *Levels not connected by the same letter are significantly different (p<0.05)

36 Effect of Primer Classification by Assay (β-actin) on Ct Ct GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) ao(dt) Primer Classification P = Primer Class Level* Mean (N) O(dT) A (36) ao(dt) A (35) GSP (β-actin) A (36) Rand A (214) Rand/O(dT) A (212) Rand/aO(dT) A (214) *Levels not connected by the same letter are significantly different (p<0.05).

37 Effect of Primer Classification by Assay (β-actin) on ΔCt delta Ct Ct Primer Class Level* Mean (N) Rand/aO(dT) A 2.34 (214) Rand/O(dT) A 2.26 (212) Rand A 2.25 (214) GSP (β-actin) A 2.19 (36) O(dT) A 2.09 (36) ao(dt) A 2.07 (35) GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) ao(dt) Primer Classification *Levels not connected by the same letter are significantly different (p<0.05). P =

38 β-glucuronidase (GUS) bases hβ-glucuronidase transcript hβ-gus 3 Total length bases 66 bases 3 Stem Structures by m-fold

39 Amplification Plot for GUS No Primer Randomers GUS, Oligo(dT), aoligo(dt), Rand + O(dT), Rand + ao(dt) No Primer, No RT

40 Effect of Primer by Assay (GUS) on Ct 40 Ct 30 12mer 12mer/AnchOligo(dT) 12mer/Oligo(dT) 15mer 15mer/AnchOligo(dT) 15mer/Oligo(dT) 18mer 18mer/AnchOligo(dT) 18mer/Oligo(dT) 21mer 21mer/AnchOligo(dT) 21mer/Oligo(dT) 6mer 6mer/AnchOligo(dT) 6mer/Oligo(dT) 9mer 9mer/AnchOligo(dT) 9mer/Oligo(dT) AnchOligo(dT) GUS NoPrimer Oligo(dT) Primer P <

41 *Levels not connected by the same letter are significantly different (p<0.05) Effect of Primer by Assay (GUS) on Ct Primer Level* Mean (N) NoPrimer A (30) 21mer B (30) 15mer B C (30) 12mer B C D (30) 6mer B C D E (30) 18mer C D E F (30) 9mer C D E F (30) 15mer/AnchOligo(dT) D E F G (30) 15mer/Oligo(dT) D E F G (29) 12mer/Oligo(dT) E F G (30) 6mer/AnchOligo(dT) E F G (29) 18mer/Oligo(dT) E F G (30) 21mer/AnchOligo(dT) E F G (30) 9mer/Oligo(dT) E F G (30) 18mer/AnchOligo(dT) E F G (30) 6mer/Oligo(dT) E F G (30) 12mer/AnchOligo(dT) F G (29) 9mer/AnchOligo(dT) F G (30) 21mer/Oligo(dT) F G (30) AnchOligo(dT) F G (29) Oligo(dT) F G (30) GUS G (27)

42 Effect of Primer by Assay (GUS) on ΔCt delta Ct mer 12mer/AnchOligo(dT) 12mer/Oligo(dT) 15mer 15mer/AnchOligo(dT) 15mer/Oligo(dT) 18mer 18mer/AnchOligo(dT) 18mer/Oligo(dT) 21mer 21mer/AnchOligo(dT) 21mer/Oligo(dT) 6mer 6mer/AnchOligo(dT) 6mer/Oligo(dT) 9mer 9mer/AnchOligo(dT) 9mer/Oligo(dT) AnchOligo(dT) GUS NoPrimer Oligo(dT) Primer P <

43 Effect of Primer by Assay (GUS) on ΔCt Primer Level* Mean (N) GUS A 4.59 (27) Oligo(dT) A 4.52 (30) 21mer/Oligo(dT) A B 4.43 (30) 9mer/AnchOligo(dT) A B 4.41 (29) AnchOligo(dT) A B 4.32 (30) 12mer/AnchOligo(dT A B 4.29 (29) ) 6mer/Oligo(dT) A B 4.25 (30) 18mer/AnchOligo(dT A B 4.22 (30) ) 9mer/Oligo(dT) A B 4.22 (30) 21mer/AnchOligo(dT ) A B C 4.08 (30) 18mer/Oligo(dT) A B C 4.07 (30) 12mer/Oligo(dT) A B C D 3.94 (30) 6mer/AnchOligo(dT) A B C D 3.92 (29) 15mer/AnchOligo(dT A B C D 3.86 (30) ) 15mer/Oligo(dT) B C D E 3.70 (29) 9mer C D E F 3.36 (30) 18mer D E F 3.22 (30) 6mer E F 2.99 (30) 12mer F G 2.59 (30) 15mer G 2.04 (30) 21mer G 1.79 (30) NoPrimer H 0.22 (30) *Levels not connected by the same letter are significantly different (p<0.05)

44 Effect of Primer Classification by Assay (GUS) on Ct Ct GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) ao(dt) Primer Classification P < Primer Class Level* Mean (N) Rand A (180) Rand/O(dT) B (179) Rand/aO(dT) B (178) ao(dt) B (29) O(dT) B (30) GSP (GUS) B (27) *Levels not connected by the same letter are significantly different (p<0.05).

45 Effect of Primer Classification By Assay (GUS) on ΔCt delta Ct Ct GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) ao(dt) Primer Classification P < Primer Class Level* Mean (N) GSP (GUS) A 4.59 (27) O(dT) A 4.52 (30) ao(dt) A 4.32 (29) Rand/aO(dT) A 4.13 (178) Rand/O(dT) A 4.10 (179) Rand B 2.67 (180) *Levels not connected by the same letter are significantly different (p<0.05).

46 TBP htata Binding Protein transcript 746 bases 5 htbp 3 Total length bases 80 bases 8 Stem Structures by m-fold

47 Amplification Plot for TBP TBP Randomers, Oligo(dT), aoligo(dt), Rand + O(dT), Rand + ao(dt) No Primer

48 Effect of Primer by Assay (TBP) on Ct 40 Ct 30 12mer 12mer/AnchOligo(dT) 12mer/Oligo(dT) 15mer 15mer/AnchOligo(dT) 15mer/Oligo(dT) 18mer 18mer/AnchOligo(dT) 18mer/Oligo(dT) 21mer 21mer/AnchOligo(dT) 21mer/Oligo(dT) 6mer 6mer/AnchOligo(dT) 6mer/Oligo(dT) 9mer 9mer/AnchOligo(dT) 9mer/Oligo(dT) AnchOligo(dT) NoPrimer Oligo(dT) TBP Primer P <

49 Effect of Primer by Assay (TBP) on Ct Primer Level* Mean (N) NoPrimer A (35) Oligo(dT) B (36) AnchOligo(dT) B C (35) 21mer B C D (36) 15mer/AnchOligo(dT) B C D E (36) 18mer/AnchOligo(dT) B C D E F (36) 18mer B C D E F (36) 21mer/AnchOligo(dT) B C D E F (36) 21mer/Oligo(dT) B C D E F (36) 15mer/Oligo(dT) B C D E F (36) 18mer/Oligo(dT) B C D E F (36) 12mer/AnchOligo(dT) B C D E F (36) 15mer B C D E F (36) 12mer/Oligo(dT) B C D E F (36) 9mer/AnchOligo(dT) B C D E F G (36) 6mer/AnchOligo(dT) C D E F G (36) 6mer/Oligo(dT) D E F G (36) 12mer E F G (36) 9mer/Oligo(dT) F G (36) 9mer G H (36) 6mer G H (36) TBP H (33) *Levels not connected by the same letter are significantly different (p<0.05)

50 Effect of Primer Classification by Assay (TBP) on Ct Ct Ct Primer Class Level* Mean (N) O(dT) A (36) ao(dt) A B (35) Rand/aO(dT) A B (216) Rand/O(dT) B C (216) Rand C (216) GSP (TBP) D (33) GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) ao(dt) Primer Classification *Levels not connected by the same letter are significantly different (p<0.05). P <

51 Effect of Primer Classification by Assay (TBP) on ΔCt delta Ct Ct GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) ao(dt) Primer Classification Primer Class Level* Mean (N) GSP (TBP) A 4.95 (33) Rand B 3.56 (216) Rand/O(dT) C 3.34 (216) Rand/aO(dT) C 3.16 (216) O(dT) D 2.57 (36) ao(dt) D 2.55 (35) *Levels not connected by the same letter are significantly different (p<0.05). P <

52 Results-By Assay β-actin There were not any significant differences between any of the primers or combinations in their ability to affect Ct or ΔCt levels GUS The effects of oligo (dt) and anchored oligo (dt) alone or in combination with a randomer on Ct or ΔCt were significant compared to the randomers alone TBP The effects of the gene specific primer, followed by the randomers alone or in combination on Ct or ΔCt were significant compared to the effects of oligo (dt) and anchored oligo (dt)

53 LOCATION? LOCATION? LOCATION?

54 Real-Time qpcr Assay Maps hβ-actin hβ-actin transcript bases 3 Total length bases 71 bases 7 Stem Structures by m-fold bases hβ-glucuronidase transcript hβ-gus 3 Total length bases 66 bases 3 Stem Structures by m-fold htata Binding Protein transcript 746 bases 5 htbp 3 Total length bases 80 bases 8 Stem Structures by m-fold

55 Conclusions Self-priming in the RT reaction can result in the generation of cdna levels similar to those levels achieved when primers are added. The use of longer randomers (15-, 18-, and 21-mers) did not perform as well as the shorter randomers (6- and 9-mers) with respect to giving lower Ct values and higher ΔCt differences. Oligo (dt) or anchored oligo (dt) appear to be more effective primers than randomers when the assays are designed closer to the 3 end of the transcript. Oligo (dt) and anchored oligo (dt) appear to be equally as effective in generating cdna for use in qpcr

56 What is the Best Priming Strategy to Use to Generate cdna for Use in qpcr? The use of randomer-oligo (dt) or randomer-anchored oligo (dt) combinations in the RT reaction appear to give universally lower Ct values and higher ΔCt differences regardless of the assay location. If the cdna will be used for only 1 gene assay, the appropriate gene-specific primer may be a better choice.

57 Nucleic Acids Research Group Timothy C. Hunter (Co-Chair) Kevin L. Knudtson (Co-Chair) Deborah S. Grove Deborah J. Hollingshead Gregory L. Shipley Katia Sol-Church William L. Taylor Kathryn S. Lilley (EB liason) University of Vermont University of Iowa Penn State University University of Pittsburgh UTHSC- Houston A. I. dupont Hospital for Children UTHSC- Memphis Cambridge University

58 NARG Study: Part II Rationale: Allow users to benchmark their in-house RT protocol using provided RNA template and primers. Goal: To find an effective primer(s) that will provide optimal cdna synthesis for use in the broadest range of qpcr assays. Real-time qrt-pcr Community performs the Reverse Transcriptase (RT) Reaction Provided RNA Primers (6-mer, 9-mer, oligo (dt) 20, mixes, GSP Protocols Questionnaire/Survey Return cdna to the NARG for qpcr