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July 2001, Volume 15, Number 7, Pages 1033-1037
Table of contents    Previous  Article  Next   [PDF]
Debate Round Table
Current recommendations for positive controls in RT-PCR assays
T Lion

Children's Cancer Research Institute, Wien, Austria

Correspondence to: T Lion, Children's Cancer Research Institute (CCRI), Kinderspitalgasse 6, 1090-Wien, Austria; Fax: 43 1 40470 437

Abstract

The choice of adequate controls for reverse transcriptase (RT-) PCR analysis has been the focus of a debate pursued in Leukemia over the past 3 years.1,2 Twenty-six authors from 15 different centers contributed to the Debate,3,45,67,89,1011,1213,1415,1617,18 and the points presented have been carefully evaluated. This survey reviews the issues discussed, and presents current options for appropriate positive controls in RT-PCR assays which are based on the views shared by the majority of participants in the Debate. It is understood, however, that the recommendations presented cannot be regarded as definitive guidelines. They reflect the present state of knowledge, and certainly need to be revisited. Leukemia (2001) 15, 1033-1037.

Keywords

RT-PCR; pseudogenes; control genes; ABL; beta2-MG; beta-actin

Introduction

Appropriate positive and negative controls are an integral part of any laboratory test. The use of RT-PCR assays has rapidly increased over the recent years, and the importance of the RT-PCR methodology both in research and diagnosis is documented by a large number of published studies relying on this technique. In general, the risk of false-positive results in PCR assays, most commonly resulting from contamination with amplified DNA products, is well appreciated by the investigators. The measures used to prevent contamination and to carefully monitor its occurrence by employing multiple negative controls9 are emphasized in most papers. By contrast, the fact that false-negative results may present a problem in a highly sensitive technique such as RT-PCR seems to be recognized to a lesser extent. This might be an explanation for the apparent lack of judicious selection of positive controls in some studies. The stimulus for initiation of the current debate was provided by the relatively frequent observation of RT-PCR controls in published papers which many investigators would regard as illegitimate. It is probably for historical reasons resulting from Northern blot analysis that researchers are tempted to use amplification of beta-actin gene fragments as positive controls in RT-PCR assays. It is surprising, however, that despite several publications indicating that this gene is not suitable as a control in RT-PCR experiments,3,19,20,21 many investigators and even companies producing RT-PCR kits still rely on this housekeeping gene as a positive control. It has been claimed that under certain conditions beta-actin might nonetheless serve as a legitimate RT-PCR control.4,22 However, when a sufficiently large number of patients are tested, beta-actin primers and experimental conditions that seem to provide valid results in most assays may turn out to be inappropriate in some instances.14 The remarkable adherence to the use of the beta-actin gene as a control23 appears quite curious in view of the possible alternatives lacking the problems associated with the use of this gene in the RT-PCR setting.

The requirements for adequate positive controls in RT-PCR assays can be discussed on the basis of the postulate that they must provide information on the availability of sufficiently intact target RNA, and permit surveillance of the reverse transcription and PCR amplification steps.

General considerations

RNA integrity

Quantification of nucleic acid preparations is commonly carried out by densitometry. This measurement, however, yields no information on the integrity of purified RNA, and it is not possible therefore to reliably quantify the actual amount of RNA available for the subsequent RT and PCR steps. If degraded RNA is present in the sample analyzed, densitometric measurement will result in overestimation of the amount of amplifiable RNA. A number of investigators recommend testing of total RNA preparations by evaluating ribosomal bands upon electrophoresis in polyacrylamide or agarose gels.6,15 Comparison with appropriate standards representing defined amounts of RNA (eg 16S- and 23S-ribosomal RNA from E. coli MRE600, Roche Diagnostics, Penzberg, Germany) permits reliable estimation of intact RNA in the sample tested. Moreover, this assay shows the occurrence of RNA degradation, and may therefore help avoid further processing of inadequate RNA preparations.

In RT-PCR assays, the most common approach to the assessment of amplifiable template RNA/cDNA is the amplification of a control gene, usually a housekeeping gene with minimal tissue- and developmental stage-specific variations.18

Due to the importance of selecting adequate genes as controls and the apparent difficulty in making appropriate choices, the main focus of this paper will be the discussion of critical issues pertaining to this problem.

RNA contamination with genomic DNA

Most of the methods currently used for isolation of RNA provide good quality RNA, but may contain variable amounts of genomic DNA (gDNA). Extraction of RNA by ultracentrifugation through a CsCl cushion yields pure RNA that is generally free of gDNA contamination,6,15 but this technique is rather laborious and its use may therefore be restricted to special applications. Since the measurement of RNA yield by densitometry does not provide any information on the presence of contaminating gDNA, the amount of RNA in the sample tested may be overestimated.

The problem of contamination with gDNA must also be considered when selecting controls expected to specifically assess the presence of amplifiable RNA. It has been demonstrated that traces of gDNA in completely degraded RNA preparations may lead to misinterpretation of the control experiment if the targeted gene does not permit distinction between gDNA- and RNA/cDNA-derived amplification products.3,24 Treatment of RNA extracts by ultraviolet irradiation25 or by RNase-free DNase may be performed in attempts to overcome this problem.4,24 Contaminating gDNA, however, may not be completely eliminated, thus permitting the generation of PCR signals even in the absence of RNA.26 Moreover, an adverse effect of the treatment on the RT-PCR yield cannot be excluded.10,13

In some instances the contamination of RNA preparations with gDNA may impair the sensitivity of RT-PCR assays. For example, the presence of gDNA has been reported to reduce the efficiency of ABL cDNA amplification, but not to the same extent as that of BCR-ABL.16 Similarly, gDNA contamination was shown to interfere with the amplification of PML-RARa cDNA, but not with the normal RARa gene transcript.16 Presence of gDNA in RNA preparations can be assessed, eg by control amplification of promoter elements which are not included in RNA transcripts4 or by amplification of a control gene sequence across a small intron which will yield products of different size depending on whether gDNA or cDNA serve as template.18 In assays in which the presence of DNA traces does not interfere with specific amplification of the RNA/cDNA target, it is only necessary to demonstrate the presence of amplifiable cDNA in the sample tested. This can be achieved by selection of an appropriate control gene sequence encompassing a large intron. If gDNA is present in the sample investigated, it will not lead to generation of a PCR product. It is necessary to ensure, however, that the control gene used has no pseudogenes in the human genome in which case gDNA and cDNA templates may give rise to similar-sized amplification products. Due to the risk of false-positive control experiments, a gene that has pseudogenes in the genome investigated should not be regarded as an adequate control in RT-PCR assays. Table 1 shows a number of genes frequently used as positive controls for amplifiable cDNA which should be avoided for this reason. For a number of other genes, the existence of pseudogenes is not known at present. Primers designed to specifically amplify mRNA of a given control gene should be tested against genomic DNA derived from the same source of cells to exclude the possibility of amplifying pseudogene sequences.14 Moreover, controls in which the RT-step is omitted are essential to exclude the possibility of homologous gene amplification,13 particularly when testing new primer combinations for amplification of a control gene.

In various studies, housekeeping genes with well-documented existence of pseudogenes, such as beta-actin (Table 1), have been used as controls for RNA/cDNA integrity. This renders negative results of RT-PCR assays questionable, because it cannot be excluded that they were attributable to RNA degradation rather than to true absence of the target transcript in the sample investigated. For beta-actin, primer combinations have been described which were claimed to be RNA-specific.22 It has been shown, however, that such primers may nevertheless result in the generation of similar-sized PCR products from DNA template in some individuals or under certain experimental conditions.14 Primer sets that do not amplify pseudogene sequences in most instances may therefore not be relied on as having absolute RNA/cDNA specificity. If for some reason, a gene with known existence of pseudogenes is selected as a control for RNA/cDNA integrity, and is tested by presumably RNA-specific primer combinations, parallel investigation of a DNA sample derived from the same patient should be performed.

RNA expression level

Suitable control genes should be expressed constitutively in a cell cycle-independent manner. It is obvious that the control gene used should not be expressed at a considerably higher level than the gene under investigation because in samples that are small or partially degraded, the amount of specific target sequences could be below the PCR detection limit, while the control gene might still show a positive amplification signal. This finding would suggest the presence of sufficient quantities of amplifiable RNA/cDNA, and could lead to false negative results. Some of the frequently used control genes such as -actin or GAPDH (Table 1) are expressed at a much higher level than most other genes,19,20,26 and therefore do not provide useful controls in most instances regardless of the presence of pseudogenes. A higher expression level of the control gene could be compensated, at least in part, by amplifying the target gene over a greater number of PCR cycles. It appears more reasonable, however, to select a control gene with a level of expression lower or equal to the gene of interest. Several authors indicated that single-step amplification of the ABL or BCR genes provides a suitable control in a variety of diagnostic PCR assays.16,18,20,27 Both genes are ubiquitously expressed at a relatively low level and neither have pseudogenes.

RNA stability and controls in quantitative PCR analysis

For quantitative RT-PCR assays, additional aspects need to be considered in the choice of control genes: (1) the transcript level of the control gene should be fairly constant and must not be affected by the disease in question;16 (2) the control gene should be assessed in a quantitative manner;27,28 and (3) the stability (degradation rate) of the control gene should correspond to that of the gene of interest.11,16 This is particularly relevant in quantitative analysis of samples which are transported to central referral laboratories and may be exposed to ambient temperature for several days during transfer. Stability of mRNA may also present a problem in samples which are frozen and stored for variable time periods prior to testing.11 The requirements for control genes in quantitative PCR assays indicated above may be difficult to meet, particularly because transcript stability of potential control genes and many target genes of interest is not known. The recent introduction of real-time quantitative PCR (RQ-PCR) approaches has greatly facilitated quantification of mRNA transcripts, and will undoubtedly provide information on the over-time stability of many gene transcripts of interest. The current SANCO Concerted Action (RQ-PCR European Network for MRD Detection in Leukemia; Europe against Cancer Program; <ifrjr.nord.univ-mrs.fr/nord_leukemia>) will reveal information on the degradation rates of various leukemia-associated fusion gene transcripts and several potential control genes. The data expected to emanate from this international collaborative study may serve as a basis for selection of appropriate controls in expression assays relying on quantitative PCR.

Internal vs external controls

Commonly, the target and the control gene are tested in separate PCR reactions using cDNA aliquots from the same sample as the template. If tube-to-tube variability presents a problem in a given setting,8 an internal control amplified in the same test tube is useful. To perform duplex or multiplex RT-PCR, the primers must be compatible and amplification products should be distinguishable in size if the subsequent analysis is performed using electrophoretic separation.18,29 Alternatively, the PCR products can be discriminated on the basis of different fluorescence signals.30 Multiplexing of PCR reactions may, however, lead to decreased sensitivity of the assay.18 A possibility to reduce this problem is the amplification of a target gene by single-tube two-round nested PCR31 in combination with a single-round amplification of a control gene in the same test tube. This approach provides the advantages of having an internal control and a highly sensitive, two-step amplification of the specific target sequence without the need to open the tube between the two rounds of PCR, as a means of reducing the risk of contamination.

Limitations of control gene amplification

It is necessary to keep in mind that successful amplification of a control gene only provides indirect information on the integrity of the target sequence itself. As long as the expression level and the stability of control gene transcripts in relation to the target transcript of interest are not known, selection of an appropriate control gene is difficult. Clearly, a single control gene may not permit reliable assessment of integrity of all target sequences of interest. It may be helpful therefore to test the integrity of an RNA sample by employing multiple control genes. This approach would permit a more comprehensive control of RNA quality in the sample analyzed, and yield more reliable information on the integrity of the transcript of interest. This notion has provided the stimulus for the establishment of multiplex PCR assays permitting co-amplification of up to four differentially expressed control genes.18,29 The data available indicate that the ability of these tests to assess gradual differences in the quality of RNA/cDNA preparations provides a useful tool for determining the expected sensitivity of RT-PCR assays.18

Positive controls for the reverse transcription (RT) and PCR steps

In addition to using amplification of a ubiquitously expressed control gene, the RT step and PCR amplification of the target transcript under investigation are commonly controlled by parallel processing of an RNA specimen containing the message of interest. For investigation of leukemia-associated fusion gene transcripts, the control RNA is commonly derived from cell lines known to express the fusion message. It is necessary to keep in mind, however, that the level of expression may be greatly elevated in cell lines. Therefore, appropriate dilutions should be used in control experiments.9,15

Concluding remarks on positive controls in RT-PCR assays

Control gene selection

In view of the diversity of target genes investigated by RT-PCR analysis and the different requirements for control genes in each assay, it will not be possible to establish universally applicable controls. Rather, selection of appropriate control genes must be performed judiciously according to the properties of the targeted RNA. The need to consider a number of critical issues when selecting genes for control experiments in RT-PCR assays may be difficult to accomplish, particularly if little information is available on the expression level and the stability of the RNA under investigation. Integrity of the RNA transcript targeted cannot be assessed with absolute certainty from control experiments testing the integrity of other gene transcripts. Nevertheless, demonstration of amplifiable transcripts from one or more reference genes should be regarded as the minimum requirement for controls assessing the integrity of RNA in the sample under investigation.

The validity of RT-PCR results in which a control gene is used that has pseudogenes in the genome investigated, must be regarded as questionable. In view of the available alternatives, the remarkable perseverance in the use of these genes (Table 1) observed in the literature is quite surprising.14,23 Some of the commonly used control genes which apparently lack pseudogenes in the human genome, and which are currently employed in different laboratories performing PCR diagnosis of leukemia-associated gene rearrangements include the Abelson gene (ABL), the BCR gene, the porphobilinogen deaminase gene (PBGD) and the beta-2-microglobulin gene (beta2-MG).7,14,16,18,29 In quantitative RT-PCR analysis of different targets, amplification of ABL or beta-2-microglobulin gene transcripts provided satisfactory controls.16,27,28,32 The results of the SANCO Concerted Action indicated above will provide information on expression levels and over-time stability of various candidate control genes, and will reveal optimal controls for quantitative RT-PCR assays.

Synopsis of important considerations resulting from the Debate Round Table

  1. Assessment of the overall quality of RNA preparations by evaluating ribosomal RNA bands after gel electrophoresis is a helpful supplement to the amplification of control genes in RT-PCR assays.
  2. Selection of control genes having processed pseudogenes in the human genome (Table 1) should be avoided. When testing the transcripts of such genes by PCR, even primer combinations that seem to be cDNA specific may result in amplification of similar-sized products from gDNA templates under certain experimental conditions or in certain individuals.
  3. Any new primer set for control gene amplification must be carefully tested against genomic DNA to ensure its RNA specificity, particularly if the existence of pseudogenes is not known. To account for the possible occurrence of variable pseudogenes in humans, primers should be tested against gDNA and RNA/cDNA derived from cells of the same individual.
  4. Successful amplification of a control gene transcript provides only indirect evidence of integrity of the target transcript of interest. Amplification of multiple control gene transcripts, eg by multiplex RT-PCR permits broader assessment of template quality, and may therefore yield more reliable information on integrity of the target transcripts.
  5. Suitable control genes should have no highly related homo- logues and/or pseudogenes in the human genome, and should be stably and ubiquitously expressed at a level which does not significantly exceed the expression level of the gene being investigated. For PCR analysis of most leukemia-associated fusion gene transcripts, eg the ABL and BCR genes appear to meet these requirements.
  6. When using control genes expressed at a considerably higher level than the gene under investigation, the control fragment should be amplified under conditions which will adequately compensate for the differences in the expression level: eg (a) appropriate dilution of template cDNA in the control experiment; (b) lower number of PCR cycles; or (c) single-round PCR when the target is amplified by two-round nested PCR. In assays for detection of most leukemia-associated fusion gene transcripts, this consideration applies to control genes such as PBGD or beta2-MG.
  7. The selection of primers for an appropriate control gene should either (a) prevent amplification of gDNA by spanning a large intron sequence; or (b) facilitate detection of contaminating gDNA in instances in which its presence may interfere with the results of the RT-PCR assay. Selection of primers spanning a short intron permits discrimination between PCR products resulting from gDNA and RNA/cDNA templates based on different-sized amplicons.
  8. In quantitative RT-PCR assays, additional requirements for selection of appropriate control genes must be considered: the expression level of the control gene has to be constant in the cell type analyzed and should not be affected by the disease under investigation. Moreover, the control gene and the target transcripts should display similar stability (ie degradation rate). This is of particular importance in situations in which samples are transferred to distant laboratories for molecular analysis. In a number of published studies on quantitative RT-PCR analysis of fusion transcripts in leukemia patients, the use of ABL and beta2-MG as control genes provided adequate results.

References

1 Kidd V, Lion T. Appropriate controls for RT-PCR. Debate round table. Leukemia 1997; 11: 871-881, MEDLINE

2 Lion T, Kidd V. Appropriate controls for RT-PCR. Debate round table. Leukemia 1998; 12: 1983-1993,

3 Lion T. Control genes in reverse transcriptase-polymerase chain reaction assays. Leukemia 1996; 10: 1527-1528, MEDLINE

4 Kidd VJ. Problematic controls for reverse transcription polymerase chain reactions (RT-PCR): an issue revisited. Leukemia 1997; 11: 873-874,

5 Lion T. Appropriate controls for RT-PCR. Leukemia 1996; 10: 1843, MEDLINE

6 Biondi A. Commentary: round-table debate on appropriate controls for RT-PCR. Leukemia 1997; 11: 875-876,

7 Cross N, Melo J. Commentary: round-table debate on appropriate controls for RT-PCR. Leukemia 1997; 11: 876,

8 Janssen LAJ, Bartram CR. Commentary: round-table debate on appropriate controls for RT-PCR. Leukemia 1997; 11: 876-877,

9 Macintyre E, Gabert J. Commentary: round-table debate on appropriate controls for RT-PCR. Leukemia 1997; 11: 877-878,

10 Mannhalter C, Mitterbauer G. Commentary: round-table debate on appropriate controls for RT-PCR. Leukemia 1997; 11: 878-879,

11 Paldi-Haris P, Földi J. Commentary: round-table debate on appropriate controls for RT-PCR. Leukemia 1997; 11: 879-880,

12 van der Reijden BA, Jansen JH. Commentary: round-table debate on appropriate controls for RT-PCR. Leukemia 1997; 11: 880,

13 El-Osta A. Commentary: round-table debate on appropriate controls for RT-PCR. Leukemia 1998; 12: 1987-1998,

14 Karlic H, Radolf M, Pfeilstöcker M. Commentary: round-table debate on appropriate controls for RT-PCR. Leukemia 1998; 12: 1988-1999,

15 Lo Coco F, Diverio D. Commentary: round-table debate on appropriate controls for RT-PCR. Leukemia 1998; 12: 1990-1991,

16 Tobal K, Liu Jin JA. Commentary: round-table debate on appropriate controls for RT-PCR. Leukemia 1998; 12: 1991-1992,

17 Vieira L, Boavida MG. Commentary: round-table debate on appropriate controls for RT-PCR. Leukemia 1998; 12: 1992,

18 Watzinger F, Lion T. Multiplex PCR for quality control of template RNA/cDNA in RT-PCR assays. Leukemia 1998; 12: 1984-1986, MEDLINE

19 Taylor JJ, Haesman PA. Control genes for for reverse transcriptase polymerase chain reaction (RT-PCR). Br J Haematol 1994; 86: 444-445, MEDLINE

20 Cross NCP, Lin F, Goldman J. Appropriate controls for reverse transcription polymerase-chain reaction (RT-PCR). Br J Haematol 1994; 87: 218 (letter), MEDLINE

21 Melo JV, Kent NS, Yan SH, Goldman JM. Controls for reverse transcriptase-polymerase chain reaction amplification of BCR-ABL transcripts. Blood 1994; 84: 3984-3986 (letter), MEDLINE

22 Soutar RL, Dillon J, Ralston SH. Control genes for reverse-transcription polymerase-chain reaction: a comparison of beta actin and glyceraldehyde phosphate dehydrogenase. Br J Haematol 1997; 97: 247-248, MEDLINE

23 Kreuzer KA, Lass U, Landt O, Nitsche A, Laser J, Ellerbrok H, Pauli G, Huhn D, Schmidt CA. Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of beta-actin transcripts as quantitative reference. Clin Chem 1999; 45: 297-300, MEDLINE

24 Dirnhofer S, Berger C, Untergasser G, Geley S, Berger P. Human beta-actin retropseudogenes interfere with RT-PCR. TIG 1995; 11: 380-381, MEDLINE

25 Sarkar G, Sommer SS. Removal of DNA contamination in polymerase chain reaction reagents by ultraviolet irradiation. In: Wu R (ed.). Methods in Enzymology. vol. 218: Academic Press: San Diego, 1993, pp 381-388.

26 Garbay B, Boue-Grabot E, Garret M. Processed pseudogenes interfere with reverse transcriptase polymerase chain reaction controls. Anal Biochem 1996; 237: 157-159, MEDLINE

27 Hochhaus A, Lin F, Reiter A, Skladny H, Mason PJ, van Rhee F, Shepherd PC, Allan NC, Hehlman R, Goldman JM, Cross NC. Quantification of residual disease in chronic myelogenous leukemia patients on interferon-alpha therapy by competitive polymerase chain reaction. Blood 1996; 87: 1549-1555, MEDLINE

28 Cross NCP, Lin F, Chase A, Bungey J, Hughes TP, Goldman JM. Competitive polymerase chain reaction to estimate the number of BCR/ABL transcripts in chronic myeloid leukemia after bone marrow transplantation. Blood 1993; 82: 1929-1936, MEDLINE

29 Mannhalter C, Koizar D, Mitterbauer G. Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA. Clin Chem Lab Med 2000; 38: 171-177, MEDLINE

30 Von Ahsen N, Oellerich M, Schutz E. Use of two reporter dyes without interference in a single-tube rapid-cycle PCR: alpha(1)-antitrypsin genotyping by multiplex real-time fluorescence PCR with the LightCycler. Clin Chem 2000; 46: 156-161, MEDLINE

31 Trka J, Divoky V, Lion T. Prevention of product carry-over by single tube two-round (ST-2R) PCR: application to BCR-ABL analysis in chronic myelogenous leukemia. Nucleic Acids Res 1995; 23: 4736-4737, MEDLINE

32 Lion T, Gaiger A, Henn T, Hörth E, Haas OA, Geissler K, Gadner H. Use of quantitative polymerase chain reaction to monitor residual disease in chronic myelogenous leukemia during treatment with interferon. Leukemia 1995; 9: 1353-1360, MEDLINE

33 Tolan DR, Niclas J, Bruce BD, Lebo RV. Evolutionary implications of the human aldolase-A, -B, -C, and -pseudogene chromosome locations. Am J Hum Genet 1987; 41: 907-924, MEDLINE

34 Moos M, Gallwitz D. Structure of two human beta-actin-related processed genes one of which is located next to a simple repetitive sequence. EMBO J 1983; 2: 757-761, MEDLINE

35 Masters JN, Yang JK, Cellini A, Attardi G. A human dihydrofolate reductase pseudogene and its relationship to the multiple forms of specific messenger RNA. J Mol Biol 1983; 167: 23-36, MEDLINE

36 Arcari P, Martinelli R, Salvatore F. Human glyceraldehyde-3-phosphate dehydrogenase pseudogenes: molecular evolution and possible mechanisms of amplification. Biochem Genet 1989; 27: 439-450, MEDLINE

37 Wells D, Bains W. Characterization of an unusual human histone H3.3 pseudogene. DNA Seq 1991; 2: 125-127, MEDLINE

38 Sellner LN, Turbett GR. The presence of a pseudogene may affect the use of HPRT as an endogenous mRNA control in RT-PCR. Mol Cell Probes 1996; 10: 481-483, MEDLINE

Tables

Table 1 Genes with known processed pseudogenes which are commonly used as RT-PCR controls

Received 21 February 2001; accepted 22 February 2001
July 2001, Volume 15, Number 7, Pages 1033-1037
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