Protocol

Quantitative Real-Time RT-PCR (qRT-PCR) of Zebrafish Transcripts: Optimization of RNA Extraction, Quality Control Considerations, and Data Analysis

¹ School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand
² LabPLUS, Auckland City Hospital, Auckland 1148, New Zealand

³Corresponding author (DonaldL{at}adhb.govt.nz).

INTRODUCTION

The zebrafish (Danio rerio) has emerged as a popular model species. The rapid development of zebrafish embryos provides opportunities for investigation of genes essential for developmental processes, the human counterparts of which might be implicated in diseases. Understanding when and where genes are expressed can facilitate greater understanding of their function, and also allow the genes to be manipulated by gene knockdown in temporally and spatially specific manners. Quantitative real-time polymerase chain reaction (qRT-PCR) is widely applied in gene expression studies. This protocol presents techniques to optimize RNA isolation from zebrafish embryos; quality assessment and the use of multiple reference genes are also emphasized. The combined use of TRIzol extraction and column-based purification is strongly recommended, because the resulting RNA is of better quality than RNA isolated using either of those methods alone. The procedure can be performed in 2 d, with individual stages taking up to 15 h to complete.

RELATED INFORMATION

This protocol is adapted from the manufacturers’ instructions for the use of TRIzol (Invitrogen) and the RNeasy Micro kit (QIAGEN). The combined method is widely used by researchers across different disciplines. However, this protocol suggests extra steps that help improve the quality and quantity of RNA isolated from zebrafish embryos. For analysis of qRT-PCR data, software such as geNorm (http://medgen.ugent.be/~jvdesomp/genorm; Vandesompele et al. 2002) and LinRegPCR (http://www.hartfaalcentrum.nl/index.php?main=files&sub=0; Ramakers et al. 2003; Ruijter et al. 2009) is available for download.

MATERIALS

Reagents

β-mercaptoethanol, 98% (Sigma M3148)

Agilent RNA 6000 Nano Kit (Agilent Technologies 5067-1511)

This kit is designed for use with Agilent Technologies’ 2100 Bioanalyzer.

Chloroform

Diethyl pyrocarbonate (DEPC)-treated H₂O

DNase, RNase-free (e.g., RNase-Free DNase Set; QIAGEN)

dNTP mix (10 mM; Invitrogen) (for reverse transcription)

Ethanol (100%, 80%, and 75%, prepared in DEPC-treated H₂O)

Glycogen, (20 mg/mL; Invitrogen)

H₂O, DNase- and RNase-free

Isopropanol

Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen)

Random primers (3 µg/µL; Invitrogen)

Dilute stock to 50 ng/µL in DEPC-treated H₂O before use.

Reagents for sequencing amplicons

Reverse transcriptase (e.g., SuperScript III; Invitrogen)

RNA Assay Kit, Quant-IT (Invitrogen) (optional; see Step 45)

This kit is designed specifically for use with the Qubit fluorometer.

RNaseZap (Ambion)

RNeasy Micro Kit (QIAGEN)

The kit includes DNase, RDD buffer, RLT buffer, RPE buffer, RW1 buffer, 1.5-mL eluate tubes, 2-mL collection tubes, and MinElute spin columns.

SDS (sodium dodecyl sulfate; 10%)

Dilute the 10% stock to 1% with DEPC-treated H₂O before use.

TRizol (TRIzol, Invitrogen)

Zebrafish embryos of the stage of development of interest

Equipment

Bioanalyzer (e.g., 2100; Agilent Technologies G2940CA)

Dry ice

Fast real-time PCR system, equipped with 384-well block module and automation accessory (e.g., 7900HT; Applied Biosystems 4329002) and software (e.g. Sequence Detection System [SDS] v2.3; Applied Biosystems)

Centrifuge, equipped for multiwell plates

Centrifuge, refrigerated, equipped for microcentrifuge tubes

Centrifuge, ventilated, for microcentrifuge tubes

Equipment for sequencing amplicons

Fluorometer, Qubit (Invitrogen Q32857) (optional; see Step 45)

Fume hood

Gloves, disposable

Homogenizer, hand-held (e.g., PRO200; PRO Scientific 01-02200)

Ice

Liquid nitrogen (optional; see Step 4.i)

Micropipettors

Optical adhesive film (e.g., MicroAmp; Applied Biosystems)

Pipette tips, barrier, 10-, 20-, 200-, and 1000-µL

Pipette tips, filter, 50-µL, PCR clean (e.g., Eppendorf 0030 003.950)

These tips are designed for use with the epMotion automated pipetting system.

Pipettes

Pipettes, transfer, single-use, 3.5-mL (Sarstedt 86.1172.001)

Pipetting system, automated (e.g., epMotion 5075 LH, 230 V; Eppendorf 5075 000.008)

Plates, 384-well (e.g., MicroAmp optical 384-well reaction plate with barcode; Applied Biosystems)

Software for data analysis (e.g., geNorm [see Step 51.iii, Step 60] and LinRegPCR [see Step 58]) (see also Related Information and Discussion)

Software for primer design (optional; see Step 50)

Spectrophotometer (e.g., NanoDrop; Thermo Scientific) (optional; see Step 45)

Thermal cycler

Timer

Tube racks, for 1.5- to 2-mL microcentrifuge tubes

Tubes, microcentrifuge, clear, 1.7-mL (e.g., Axygen MCT-175-C)

Tubes, PCR, thin-wall, flat-cap, 0.2-mL (e.g., MAXYMum Recovery; Axygen PCR-02-L-C)

Tubes, RNase-free, nonstick, 1.5-mL (e.g., Ambion AM12450)

METHOD

To minimize RNase contamination, clean work surfaces with RNaseZap thoroughly. Open tubes facing away from the operator, and avoid breathing into tubes. Dispense aliquots of solutions into sterile tubes; dispose of tubes after each use. Change gloves frequently, and unless specified in the protocol, keep RNA on ice as much as possible.

Preparation

1. Soak the probe of the homogenizer in 1% SDS overnight.

2. Wash the 1.7-mL microcentrifuge tubes to be used for homogenization:

i. Wash once with 1 mL of DEPC-treated H₂O.

ii. Wash once with 1 mL of 75% ethanol.

iii. Wash once with 800 µL of TRIzol.

Sample Collection

Embryo dechorionation is not mandatory, but can be performed if desired.

3. Dispense 20-50 embryos into a washed 1.7-mL microcentrifuge tube. Remove excess liquid using a disposable transfer pipette.

4. Euthanize the embryos using one of the following methods:
The effects of anesthetics (e.g., Tricaine) on the efficiency of the extraction protocol and downstream amplification of transcripts are not known at present.

Freezing:

i. This is the preferred method of euthanasia.
Immerse the tube in liquid nitrogen.
Samples can be stored at -80°C.

ii. Proceed to Step 5.

TRIzol extraction:

iii. Prechill TRIzol on ice.

iv. Add 1 mL of TRIzol to each tube.

v. Proceed immediately to Step 5 and then Step 8.

Homogenization

5. Wash the homogenization probe (10-sec each wash):

i. Wash twice with DEPC-treated H₂O.

ii. Wash once with RNaseZap.

iii. Wash once with DEPC-treated H₂O.

iv. Wash once with 75% ethanol.

v. Wash once with TRIzol.

6. Transfer frozen embryos from -80°C storage to the bench on dry ice.

7. Add 1 mL of TRIzol to a 1.7-mL microcentrifuge tube containing frozen embryos.

8. Homogenize the sample for 30 sec (Fig. 1 ).

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Figure 1. Homogenizer setup in a portable fume hood.

9. Incubate the tube on dry ice for 30 sec.
Allow the homogenizer to cool down to prevent overheating of the sample.

10. Homogenize the sample for 30 sec.

11. Incubate the sample for 5 min at room temperature.
Repeat Steps 8-11 for each sample. Between samples, wash the probe, 10 sec each wash, once with 75% ethanol, once with DEPC-treated H₂O, and once with TRIzol.

12. Place samples on dry ice for same-day use, or store for later use at -80°C.

13. After homogenizing all samples, clean the probe (10 sec each wash):

i. Wash once with 75% ethanol.

ii. Wash once with DEPC-treated H₂O.

iii. Wash once with 75% ethanol.

iv. Wash once with DEPC-treated H₂O.

RNA Isolation with TRIzol

14. Thaw frozen homogenized samples on ice.

15. Add 200 µL of chloroform to each sample. Shake vigorously for 15 sec. Incubate for 3 min at room temperature.

16. Centrifuge at 10,000g for 15 min at 4°C.

17. Transfer 550 µL of the aqueous phase to an RNase-free tube.

18. Add 1 µL of 20-mg/mL glycogen to each sample. Add 550 µL of isopropanol.
For young embryos or for a small number of embryos, adding glycogen increases RNA yields significantly.

19. Incubate for 10 min at room temperature.

20. Centrifuge at 10,000g for 10 min at 4°C. Remove the supernatant.

21. Wash the pellet once with 1 mL of 75% ethanol. Centrifuge at 10,000g for 5 min at 4°C.
While centrifuging, prepare 1.5-mL "waste" microcentrifuge tubes.

22. Decant the supernatant into the "waste" tubes.
If the pellet dislodges from the tube, recentrifuge to recover the pellet.

23. Recentrifuge the sample tubes at 10,000g for 5 min at 4°C.

24. Carefully remove the remaining supernatant using a 20-µL micropipettor (set the volume to 20 µL).

25. Resuspend each pellet in 100 µL of DEPC-treated H₂O.
Keep samples on ice until column purification.

RNA Purification Using Columns and DNase Treatment

Use the RNeasy Micro Kit (QIAGEN).

Perform all centrifugations in a nonrefrigerated microcentrifuge.

26. Add β-mercaptoethanol to the RLT buffer to a final concentration of 1%. Add 350 µL of the RLT buffer to 100 µL of the crude RNA solution (from Step 25).

27. Add 250 µL of 100% ethanol to the diluted RNA. Do not centrifuge. Pipette up and down three times.

28. Apply 700 µL of the sample to an RNeasy MinElute spin column in a 2-mL collection tube. Close the tube gently. Centrifuge at 8000g for 45 sec.

29. Transfer the spin column to a new 2-mL collection tube.

30. Add 700 µL of RW1 buffer. Centrifuge at 8000g for 45 sec.

31. Discard the supernatant. Reserve the collection tube.

32. For each column, mix 70 µL of RDD buffer and 10 µL of DNase. Pipette up and down gently to mix.

33. Add 80 µL of the DNase mix to the column. Incubate for 30 min.
Although the QIAGEN RNeasy Micro Kit manual suggests a 15-min digestion, a 30-min incubation is more effective, without compromising RNA integrity.

34. Add 350 µL of RW1 buffer to the column. Centrifuge at 8000g for 45 sec.

35. Transfer the spin column to a new 2-mL collection tube.

36. Pipet 500 µL of RPE Buffer onto the spin column. Incubate for 5 min.

37. Close the tube gently. Centrifuge at 8000g for 45 sec to wash the column.

38. Discard the flowthrough. Reuse the collection tube.

39. Repeat Steps 36-38 twice more, but perform the third RPE wash without the 5-min incubation.
The 5-min RPE incubation should minimize salt contamination, and can therefore improve the A₂₆₀/A₂₃₀ ratio on a spectrophotometer reading.

40. Add 500 µL of 80% ethanol to the column. Close the tube gently. Centrifuge at 8000g for 2 min to dry the silica-gel membrane.

41. Transfer the column to a new 1.7-mL microcentrifuge tube (not supplied in the kit). Open the cap. Centrifuge at 8000g for 5 min.

42. While centrifuging, label 1.5-mL eluate tubes (provided with the kit).

43. Transfer the silica column to a labeled 1.5-mL tube. Add 12 µL of DEPC-treated H₂O. Close the tube. Incubate for 1 min.

44. Centrifuge at 10,000g for 1 min to elute.

RNA Analysis

45. Determine the RNA concentration:
If the expected RNA concentration is <50 ng/µL, use a fluorometer instead of a spectrophotometer.
See Troubleshooting.

For NanoDrop spectrophotometer readings:

i. Polish the paddle very well with DEPC-treated H₂O.

ii. Use as a blank the same DEPC-treated H₂O used for eluting RNA (see Step 43).

iii. Pipette 1 µL of RNA into the spectrophotometer. Measure absorbance at 230, 260, and 280 nm.

iv. Record the RNA concentration in ng/µL, and as A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios.
For more information on spectrophotometric methods, see Quantitation of DNA and RNA (Barbas et al. 2007).
See Troubleshooting.

For fluorometric quantitation with RNA-specific dye:

v. Measure the RNA concentration using a Qubit fluorometer and a Quant-IT RNA assay kit according to the manufacturer’s instructions.
Using this system, concentrations can be determined using as little as 1 µL of RNA.

46. Determine RNA integrity using a bioanalyzer according to the manufacturer’s instructions.
Perform measurements as quickly as possible to avoid RNase contamination. Only use samples with an RNA integrity number (RIN) 7.5 for qRT-PCR or with RIN 8 for microarrays (Fig. 2 ).

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Figure 2. Bioanalyzer results. (A) Intact RNA (RIN = 10); (B) slightly degraded RNA (RIN = 8.3); (C) moderately degraded RNA (obtained from an unoptimized tissue panel set) (RIN = 6.3); (D) completely degraded RNA (obtained from an unoptimized tissue panel set) (RIN not available). The optimized extraction method should never produce RNA samples such as those in panels C and D; such samples likely are contaminated with RNase. (E) A gel-like image of bioanalyzer results for a developmental time course series. (Lane 1) 8 hours post-fertilization (hpf); (lane 2) 26 hpf; (lane 3) 30 hpf; (lane 4) 48 hpf; (lane 5) 72 hpf; (lane 6) 76 hpf; (lane 7) 98 hpf; (lane 8) 126 hpf. The RNA samples isolated at these time points all have RIN 8. RNA profiles from earlier time points show a high molecular form (i.e., >28S), relative to later time points. Only RNA with RIN of 7.5 should be used for qRT-PCR.

See Troubleshooting.

Reverse Transcription

Methods for generating cDNA from mRNA can be found in Real-Time RT-PCR: cDNA Synthesis (Kusser et al. 2006), Amplification of cDNA Generated by Reverse Transcription of mRNA (Sambrook and Russell 2006), and cDNA Synthesis and Real-Time PCR Using RNA from Laser-Captured Cells (Morimoto et al. 2006). cDNA can be stored at -20°C.

47. Calculate the volume of RNA eluate (from Step 44) equivalent to 1 µg of RNA.

48. Add 1 µg of RNA to 20-µL reactions performed in 0.2-mL PCR tubes.

49. Perform reverse transcription according to the manufacturer’s instructions for the reverse transcriptase of choice.
It is important to include a reaction without reverse transcriptase to assess the level of genomic DNA contamination in cDNA amplification by quantitative PCR (see Step 53).

Quantitative Real-Time PCR

50. Design primers:
Manual design of primers and/or probes should follow general guidelines (Malnati et al. 2008). The following parameters are recommended for use with the SYBR Green system. Alternatively, for probe-based chemistry, some manufacturers (e.g., TaqMan Custom Assays, Applied Biosystems) offer design services free of charge.

i. Use freely available primer design software (e.g., Primer3; http://fokker.wi.mit.edu/primer3/input.htm) to identify suitable primer sets according to the following criteria:
Amplicon length = 80-150 bp
Primer length = 18-25 bp
GC content = 45%-55%
Primer T_m = 59°C-62°C
Max self-complementarity = 3
Max 3' self-complementarity = 3
Max 3' stability = 7
Max poly-X = 3
Objective function penalty weights for primers:
Self-complementarity = 1
3' self-complementarity = 1

ii. If exon-intron structures are known for the genes of interest, design primers such that, for each pair of primers, at least one primer overlaps an exon boundary. If this is not possible, primer pairs should span an intron.

iii. To evaluate suitable primer sets for qRT-PCR, determine the amplification efficiency by amplifying a dilution series of reference cDNA/plasmids. Plot the Ct values against the log concentration of the cDNA/plasmids.
The amplification efficiency can be determined from the slope of the plot.

iv. Sequence the amplicons to ensure specificity.
Gel electrophoresis can also be used to examine the specificity of amplification.

51. Select reference genes:

i. Evaluate the reference genes for each experimental condition to identify a suitable reference panel (Vandesompele et al. 2002).
A panel of nine genes can comprise a reference set for developmental time course studies in zebrafish (Tang et al. 2007).

ii. Select the number of reference genes to be included in each qRT-PCR run.
The use of at least three reference genes (e.g., Rpl13, Ef1, and β-actin) is recommended for the assessment of gene expression in a developmental time course.

iii. Use geNorm to determine the most stable reference genes (usually two).
The software takes the geometric mean of quantities to derive a normalization factor for each sample.
See Troubleshooting.

52. Dilute cDNA (from Step 49) 1:5 to 1:20 (depending on the level of expression of the gene of interest).

53. Set up qRT-PCR reactions using the following:
1X Platinum SYBR Green qPCR SuperMix-UDG (containing ROX reference dye at a final concentration of 50 nM)
0.3 µM each of the forward and reverse primers
2 µL of template RNA
2 µL of diluted cDNA
RNase- and DNase-free H₂O to a final volume of 10 µL
Perform reactions using H₂O-only and cDNA reactions generated without added reverse transcriptase (from Step 49) as controls to ensure that the reactions are free of contamination from genomic DNA. Optimize the concentrations of forward and reverse primers. For example, test a combination of different concentrations of both primers to obtain a single amplicon free of primer dimer artifacts.

54. Pipette reactions into a 384-well optical plate using an automated pipetting system.
Use optical adhesive film to reduce well-to-well contamination and sample evaporation.
The SYBR Green qPCR mix is quite viscous; set the "Liquid type" to "Glycerol."

55. Assay samples in triplicate using a fast real-time PCR system (Fig. 3 ):

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Figure 3. Pipetting accuracy can be determined by examining triplicate reactions using the same cDNA.

i. Perform 40 amplification cycles, with each cycle consisting of 15 sec at 95°C, followed by 1 min at 60°C.

ii. Include melt curve analyses in each assay. For example, if using the 7900HT fast real-time PCR system, add a "Dissociation Stage" at the end of the amplification cycles (e.g., a cycle of 15 sec at 95°C, 15 sec at 60°C, and 15 sec at 95°C).

Data Analysis

56. Analyze gene expression using amplification and dissociation curves (Fig. 4 ) generated by the SDS v2.3 software.

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Figure 4. Melt curve analysis. The two curves are the result of amplification of different cDNA samples using the same primer set in different cDNA samples: (A) A double peak might be caused by nonspecific amplification, as demonstrated by the real-time products seen on the 2% agarose gel (right). In this case, the double peak might be the result of the presence of alternatively spliced exons. (B) Amplification results from target cDNA. Only one band appears in the gel (right). An optimal primer set should always produce a melt curve profile such as that shown in B.

See Troubleshooting.

57. Remove outliers from the data.

58. Export fluorescence data to LinRegPCR. Calculate amplification efficiency:
Amplification efficiencies from individual reactions obtained from LinRegPCR can be used, or an average of amplification efficiencies for the same primer set can be determined and then used in calculating quantities (see Step 59).
See Troubleshooting.

i. Plot the log (i.e., absolute fluorescence) as the y-axis and the cycle number as the x-axis.
There is a "window of linearity" from which LinRegPCR (Ramakers et al. 2003) selects the points with the best correlation coefficient.

ii. Calculate the efficiency from the slope.
The ideal value is 2, which means that the PCR product/fluorescence doubles with each cycle.

59. Convert the C_t values for target and reference genes into raw quantities using the formula:
Q = E^Ct = E^{(Min Ct - Sample Ct)}
where "Q" is the sample quantity (relative to the sample with the highest expression), "E" is the amplification efficiency (2 = 100% efficiency), "Min Ct" equals the lowest Ct value, and "Sample Ct" is the Ct value of the sample with the highest expression.

60. Input quantities of reference genes into geNorm. Perform stepwise exclusion of the least stable gene until the two most stable genes are left.

61. Calculate normalization factors for each sample.
"Sample" can be different time points, control, or treatments.

62. Calculate normalized quantities by dividing raw quantities by normalization factors.

TROUBLESHOOTING

Problem: RNA yield is low.

[Step 45]

Solution: Consider the following:

1. Ensure that all reagents and equipment and the work environment are RNase-free.

2. Increase the number of embryos homogenized in each sample.

3. Make sure that the samples are thoroughly homogenized; no lumps should be visible after homogenization.

4. If necessary, increase the homogenization time.

Problem: The A₂₆₀/A₂₈₀ ratio is <2, indicating possible protein contamination.

[Step 45]

Solution: Avoid taking the interface when removing the aqueous phase during TRIzol extraction.

Problem: The RNA is degraded.

[Step 46]

Solution: Consider the following:

1. Only use RNase-free equipment such as microcentrifuge tubes and pipette tips.

2. Be sure to clean the work area with RNaseZap.

3. Change gloves regularly.

Problem: The reference genes are unstable.

[Step 51]

Solution: Revalidate another set of reference genes for each experimental condition. Use appropriate software to assess the stability of the new set of reference genes.

Problem: There is no amplification.

[Step 56]

Solution: Consider the following:

1. Dilute cDNA from reverse transcriptions for use in qPCR reaction. Some components from reverse transcription can inhibit PCR.

2. Extracted RNA can contain impurities. Repeat the extraction. Run regular PCRs and perform gel electrophoresis to determine if amplification is evident. If not, redesign primers.

Problem: There is genomic DNA contamination.

[Step 56]

Solution: Consider the following:

1. Use more TRIzol or avoid the interface while removing aqueous phase.

2. Use RNase-free DNase such as the QIAGEN RNase-Free DNase Set or any other kits that are compatible with reverse transcription and qRT-PCR.

Problem: Amplification efficiency is poor.

[Step 58]

Solution: Consider the following:

1. Try different primer concentration combinations.

2. Optimize the magnesium concentration for each primer set.

3. Design new primers. Further information is available in PCR Primer Design (Apte and Daniel 2009) and Optimization and Troubleshooting in PCR (Roux 2009).

DISCUSSION

Use of this protocol routinely produces high-quality RNA from zebrafish embryos obtained over a broad range of time points (4.5-126 hours post-fertilization [hpf]). For example, the RNA yield from 25 6-hpf-embryos is ~4.5 µg, which is sufficient for amplifying at least four different reference genes and four genes of interest in triplicate reactions containing 20 ng of input cDNA.

In terms of RNA quality assessment, spectrophotometer readings are important: The A₂₆₀/A₂₈₀ ratio gives an indication of protein contamination. Do not use any RNA in which the A₂₆₀/A₂₈₀ is <2. With respect to RNA integrity, this protocol routinely achieves good RIN (8). Although no direct comparisons were made between the hybrid approach described here and the use of TRIzol extraction alone, residual contaminants and small RNAs (5S and tRNA) are a common problem in RNA isolated using single-step organic extraction protocols. These contaminants and small RNAs can affect downstream processing (http://www.ambion.com/techlib/tn/112/10.html); combining TRIzol and column-based purification strategies reduces such downstream problems. Although TRIzol or column purification alone can yield RNA with equivalent RINs, the A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ ratios are lower than those achieved using the combined method.

The RNA isolation and purification procedures described in this protocol can easily be adapted to extract RNA from various tissues (e.g., eyes, kidney, heart, spleen, liver, intestine, testis, ovary, brain, gill, skin, and muscle) from adult and juvenile zebrafish. If pooling is required, the use of RNALater (Ambion) is strongly recommended. The procedure routinely produces RINs of at least 7 for a tissue panel, with RIN of 8 for the majority of tissues. The RNA isolated using this protocol is also suitable for microarray studies.

With well-designed primer sets and optimized qRT-PCR assays, the data analysis should be straightforward. If SYBR Green chemistry is used, a good primer set should only have one single peak in the dissociation curve analysis. Close Ct values among technical replicates indicate accurate pipetting by the operator. The use of multiple reference genes is highly recommended; this alleviates the danger of relying on a single reference gene, where the stability of the reference gene can vary. Other software, such as BestKeeper (Pfaffl et al. 2004) or NormFinder (Andersen et al. 2004), can be used instead of using geNorm. For estimating amplification efficiency, models other than LinRegPCR are also available (Liu et al. 2002a,b; Tichopad et al. 2004). Investigators should evaluate different models for calculating fold change/relative expression of their genes of interest. Alternative models, such as the 2^-C_T method (Livak and Schmittgen 2001) or the Pfaffl method (Pfaffl 2001), also can be applied.

REFERENCES

Andersen CL, Jensen JL, Ørntoft TF. 2004. Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64: 5245–5250.[Abstract/Free Full Text]

Apte A, Daniel S. 2009. PCR primer design. Cold Spring Harb Protoc doi: 10.1101/pdb.ip65.[Abstract/Free Full Text]

Barbas CF III, Burton DR, Scott JK, Silverman GJ. 2007. Quantitation of DNA and RNA. Cold Spring Harb Protoc doi: 10.1101/pdb.ip47.[Abstract/Free Full Text]

Kusser W, Javorschi S, Gleeson MA. 2006. Real-time RT-PCR: cDNA synthesis. Cold Spring Harb Protoc doi: 10.1101/pdb.prot4114.[Abstract/Free Full Text]

Liu W, Saint DA. 2002a. A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal Biochem 302: 52–59.[Medline]

Liu W, Saint DA. 2002b. Validation of a quantitative method for real time PCR kinetics. Biochem Biophys Res Commun 294: 347–353.[Medline]

Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-C_T method. Methods 25: 402–408.[Medline]

Malnati MS, Scarlatti G, Gatto F, Salvatori F, Cassina G, Rutigliano T, Volpi R, Lusso P. 2008. A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load. Nat Protoc 3: 1240–1248.[Medline]

Morimoto M, Morimoto M, Whitmire J, Star RA, Urban JF Jr, Gause WC. 2006. cDNA synthesis and real-time PCR using RNA from laser-captured cells. Cold Spring Harb Protoc doi: 10.1101/pdb.prot4108.[Free Full Text]

Pfaffl MW. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45. doi: 10.1093/nar/29.9.e45.[Abstract/Free Full Text]

Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol Lett 26: 509–515.[Medline]

Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM. 2003. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339: 62–66.[Medline]

Roux KH. 2009. Optimization and troubleshooting in PCR. Cold Spring Harb Protoc doi: 10.1101/pdb.ip66.[Abstract/Free Full Text]

Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, Moorman AFM. 2009. Amplification efficiency: Linking baseline and bias in the analysis of quantitative PCR data. Nucl Acids Res 37: e45. doi: 10.1093/nar/gkp045.[Abstract/Free Full Text]

Sambrook J, Russell DW. 2006. Amplification of cDNA generated by reverse transcription of mRNA. Cold Spring Harb Protoc doi: 10.1101/pdb.prot3837.[Free Full Text]

Tang R, Dodd A, Lai D, McNabb WC, Love DR. 2007. Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization. Acta Biochim Biophys Sin (Shanghai) 39: 384–390.[Medline]

Tichopad A, Didier A, Pfaffl MW. 2004. Inhibition of real-time RT-PCR quantification due to tissue-specific contaminants. Mol Cell Probes 18: 45–50.[Medline]

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: research0034.1–research0034.11.