1. Advanced Environmental Biotechnology II
Week 06 - expression analysis of functional genes

2. 5 RT-PCR and mRNA expression analysis of functional genes
Balbina Nogales

3. 5.1 Introduction
Microbial communities are essential for the functioning of ecosystems.
Their activity is regulated by environmental factors and depends on the activities of their individual members and populations and interactions amongst them.
They are complex, metabolically flexible and highly adaptable to changing environmental conditions, with their function finely regulated at the molecular level.

4. The analysis of microbial processes in the environment an important challenge for microbial ecologists.
Ecophysiological approaches rely on the detection of products resulting from a particular process and the measurement of transformation rates.
Now molecular biology techniques, such as the analysis of gene expression via detection of mRNA after reverse transcription-polymerase chain reaction (RT-PCR), can show microbial function.

5. Reverse transcription occurs in the cytoplasm of cells infected by viruses. In this process, viral ssRNA is transcribed by the viral reverse transcriptase (RT) into double stranded DNA.
Reverse transcription takes place in 3'→5' direction. tRNA ("cloverleaf") hybridizes to PBS and provides -OH group for initiation of reverse transcription.

6. 1) Strong stop complementary DNA (cDNA) is formed.
2) Template in RNA:DNA hybrid is degraded by RNase H domain of reverse transcriptase
3) DNA:tRNA is transferred to the 3'-end of the template (synthesis "jumps").

7. 4) First strand synthesis takes place.
5) The rest of viral ssRNA is degraded by RNase H, except for PP site.
6) Synthesis of second strand of ssDNA is initiated from the 3'-end of the template. tRNA is necessary to synthesis of complementary PBS.

8. 7) tRNA is degraded
8) After another "jump", PBS from the second strand hybridizes with the complementary PBS on the first strand.
9) Synthesis of both strands is completed by the DNAP function of reverse transcriptase.

9. Both dsDNA ends have U3-R-U5 sequences, so called long terminal repeat sequences (3'LTR and 5'LTR, respectively). LTRs mediate integration of the retroviral DNA into another region of the host genome.

13. Since prokaryotic gene expression is a finely regulated process, detection of transcripts for a given gene constitutes significant evidence of the occurrence of a given biological process within the environment. Recent years have seen a significant increase in the number of studies reporting the analysis of microbial gene expression by RT-PCR in environmental systems.

14. Since prokaryotic gene expression is a finely regulated process, detection of transcripts for a given gene constitutes significant evidence of the occurrence of a given biological process within the environment. Recent years have seen a significant increase in the number of studies reporting the analysis of microbial gene expression by RT-PCR in environmental systems.

15. Most are in three main groups.
(i) analysis of gene expression in pathogenic bacteria, e.g. Staphylococcus aureus and Helicobacter pylori;
(ii) detection and analysis of expression of genes involved in biogeochemical processes eg methanotrophy, nitrogen fixation, nitrification, denitrification and carbon fixation;
(iii) investigation of the expression of genes in the biodegradation of environmental pollutants, eg aromatic hydrocarbons.

16. 5.2 Advantages and limitations of the RT-PCR analysis of bacterial functional genes
Several environmentally relevant questions can be approached by using RT-PCR-based techniques as shown in Figure 5.1.

18. Figure 5.1
Schematic view of the different RT-PCR approaches that can be applied to gene expression analysis in environmental studies.

19. RT-PCR can be used to detect transcription of a gene of interest and to determine the diversity of the transcripts being expressed in the environment. The individual microorganisms in which specific gene expression is occurring can be identified by in situ RT-PCR techniques, and these organisms can be enumerated and their spatial distribution determined. Thirdly, the effect of environmental parameters in gene expression can be explored by quantitative RT-PCR and by using techniques for global gene expression analysis such as RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) or differential display (DD), which allow for the analysis of modulation of gene expression in response to changing environmental conditions.

20. Techniques known in general as RNA fingerprinting include differential display and RNA fingerprinting by arbitrary primed PCR (RAP-PCR). Both methods are based on PCR amplification of random subsets of genes from two or more RNA samples. The first step of either is to reverse-transcribe random subsets of mRNA to cDNA.
In differential display, this is done using an anchored primer, which is typically a polyT oligonucleotide with one or two additional bases (e.g., T12AC). In contrast, RAP-PCR uses arbitrary primers in reverse transcription. These primers are typically 10 bp in length and may anneal to complementary sequence and prime reverse transcription from any point along an RNA transcript.

21. Differential display.
Total RNA is extracted from two experimental samples, 1 and 2. An anchored primer, such as T12G, is used to reverse-transcribe a subset of the mRNA to cDNA.
Random fragments of the cDNA transcripts are amplified in duplicate PCR reactions using different combinations of forward and reverse primers.
The resulting PCR products are compared to identify gene fragments expressed in sample 1 but not 2 (iii), expressed in 2 but not 1 (ii), and expressed in both 1 and 2 but at different intensities (I).
from: D. E. Moody (2001) Genomics techniques: An overview of methods for the study of gene expression J Anim Sci :E128-E135. jas.fass.org/cgi/reprint/79/E-Suppl/E128.pdf

22. RT-PCR methods are not intrusive (no incubation of samples, nor addition of substrates required).
RT-PCR-based analysis of mRNA is also culture independent, sensitive, specific, rapid, reproducible and can be adapted to high-throughput systems when the analysis of many samples is required.

23. But the analysis of gene transcription by RT-PCR has important methodological limitations.
Firstly, prior knowledge of the sequence of genes of interest is a prerequisite of such analysis to enable the design of primers to allow amplification of specific RT-PCR products.
Some of the limitations of RT-PCR techniques are more methodological, such as the quantity, quality and stability of the RNA extract to be used in the reaction.
RT-PCR amplification from environmental samples would be limited by difficulties in the RNA extraction and by the presence of inhibitory substances in the extracts which will interfere with the RT-PCR reaction.

24. Finally, since RT-PCR is highly sensitive, controls to ensure that amplification products are not derived from contaminating DNA need to be performed with every RNA extract used in RT-PCR reactions. Typically, RNA extracts are treated with RNase-free DNase and used in PCR reactions without a prior RT reaction (no-RT control).

25. 5.3 The RT-PCR reaction
RT-PCR (19) consist of two sequential steps, namely a reverse transcription reaction (RT) in which a complementary DNA molecule (cDNA) is generated from an RNA template by extension of an oligonucleotide primer due to the action of a reverse transcriptase, and a subsequent PCR amplification reaction in which the cDNA is amplified exponentially by a thermostable DNA polymerase. Both reactions can be performed separately or sequentially in the same tube. Most suppliers of molecular biology products maintain excellent web pages with useful technical information on RT-PCR procedures (Table 5.1).

26. 5.3.1 The RNA template
It is important to have high quality RNA, i.e. not degraded and free from DNA and ribonuclease contamination, or substances inhibitory to enzymatic reactions.
Prokaryotic mRNA is a labile molecule with a short half-life.
Considerable care is needed to avoid RNA degradation.
RT-PCR amplification of prokaryotic mRNA is usually performed using total RNA extracts that also contain the more abundant RNA fractions: ribosomal (rRNA) and transfer (tRNA) RNA.

27. 5.3.2 Primers for reverse transcription
Three types of primers can be used:
(i) specific primers that anneal exclusively to the mRNA of interest;
(ii) random hexanucleotides that anneal randomly to any RNA molecule (including rRNA) present in the extract;
(iii) oligo (dT) primers that bind to poly(A) tails at the 3′-end of mRNAs (rarely used for prokaryotes).
The objective is to obtain a high proportion of cDNAs complementary to the target RNA and with the maximum possible length.
The choice would in most cases be the use of specific primers for the RT reaction.
Random hexameric primers leads to the transcription of non-coding RNA fractions in addition to the mRNA fraction, although it offers advantages when the product of a single RT reaction is to be used in several PCR reactions using different primer sets.

28. 5.3.3 Reverse transcriptases
There are two reverse transcriptases of viral origin, the avian myeloblastosis virus reverse transcriptase (AMV) and the Moloney murine leukaemia virus reverse transcriptase (M-MLV), and two of bacterial origin, C. therm polymerase (from Carboxydothermus hydrogenoformans) and Tth DNA polymerase (a recombinant DNA polymerase derived from Thermus thermophilus).
Both bacterial reverse transcriptases are thermostable enzymes, thereby enabling the reaction to be performed at high temperatures and thus providing optimal conditions for primer annealing and reducing the effects of secondary structure in the template.

29. Bacterial reverse transcriptases have special features that have made them the polymerase of choice for certain applications. Tth DNA polymerase has the ability to perform both RT and PCR amplification in the presence of manganese or magnesium ions, respectively and forms the basis of the one-step one-enzyme RT-PCR protocols. C. therm polymerase is the only reverse transcriptase with an associated 3′ to 5′ proofreading activity, which results in an increased fidelity.

30. 5.3.4 One-step and two-step RT-PCR protocols
‘one-step procedure’ (also called ‘continuous’)
‘two-step procedure’ (or ‘uncoupled’)

31. In the one-step procedure, the RT and the PCR reactions take place sequentially in the same tube, in the presence of a unique buffer using a specific primer set, therefore no additional reagents need to be added during the reaction. One-step RT-PCR can be carried out using a single enzyme (Tth DNA polymerase), referred to as ‘one-enzyme’ protocols, or by using commercially available combinations of reverse transcriptase plus thermostable DNA polymerase (‘two-enzyme one-step protocols’). The one-step procedure requires less manipulation, reducing pipetting errors, time and minimizing the risk of contamination.

32. In the two-step procedure, RT and PCR reactions are done sequentially, but separately, in the optimal reaction conditions for each enzyme because a two-buffer system is used. The RT reaction can be performed with specific primers, random hexanucleotides or oligo (dT) (except when using Tth polymerase which requires the use of specific primers).
After the RT reaction is completed, PCR reagents can be added to the tube (‘one-tube system’) or an aliquot of the RT reaction is transferred to another tube and a PCR reaction with specific primers is performed (‘two-tube system’).
The two-step procedure, especially the two-tube system, provides greatest flexibility since the cDNA can be used in several PCR reactions using different primer sets. It does require more manipulation, increasing the likelihood of contamination.

33. Sensitivity seems to be greatest using the one-step two-enzyme RT-PCR protocol, followed by the two-step two-enzyme protocol with lowest sensitivities reported using the one-step one-enzyme (Tth polymerase) protocol (24). Factors other than sensitivity may also dictate the choice of RT-PCR protocol including requirements related to the product yield required, reaction specificity, or the number of samples to be processed.

34. 5.4 Quantitative RT-PCR
Quantitative RT-PCR methods have been developed for the quantification of steady-state mRNA levels. Quantification can be performed after the reaction is completed (end-point measurement) or during the course of the reaction (real-time or kinetic).
In some cases absolute quantification of the number of target copies per specific unit is performed, but most often quantitative RT-PCR is used to quantify changes in the expression of a specific gene after a treatment by comparison to a reference sample such as an untreated control (relative quantification).

35. The most frequently used quantitative RT-PCR are competitive RT-PCR and real-time RT-PCR. Both techniques are reproducible and have comparable sensitivity.
Competitive RT-PCR is based on the co-amplification of the target RNA and known amounts of a standard RNA (the competitor) that is designed to be amplified with the same primers and with the same efficiency as the target but differs in length or contains a mutation that allows its differentiation from the target.
Real-time RT-PCR, product synthesis is measured in each cycle during the exponential phase of PCR by measuring an increase in fluorescence as the reaction proceeds. Examples of quantitative analyses of gene expression in environmental samples are the quantification of ribulose-1,5-biphosphate carboxylase/oxygenase, rbcL, transcripts in diatom cultures and marine samples and the quantification of ammonia monooxygenase, amoA, transcripts in a biofilm.

36. 5.5 Analysis of global gene expression
Genome-wide screening of mRNA transcripts, produced under different environmental conditions, can be compared in order to determine genes that are differentially expressed (induced or repressed) under varying conditions. Several methods are available for analyzing global gene expression in prokaryotes including differential display (DD) and RAP-PCR. The basic principle of DD and RAP-PCR is the generation of a collection of cDNAs using short oligonucleotides that in theory covers the whole genome. The cDNAs are subsequently amplified by PCR and the products separated on polyacrylamide or agarose gels. Comparison of the band patterns allows for the detection of differentially expressed genes.

37. Band patterns are checked by other techniques such as quantitative RT-PCR, as the frequency of false positives generated by DD or RAP-PCR may be high.
The DD method uses anchored oligo (dT) primers for the RT reaction, followed by PCR amplification using the same anchored oligo (dT) primers and short arbitrary primers.
RAP-PCR uses arbitrary primers only, both for the RT and PCR reaction, so has been used most to analyze prokaryotic mRNAs.

38. In studies of prokaryotic systems, several different strategies with respect to the type of primers used for DD and RAP-PCR have been used.
Primers based on the calculation of oligomer frequency distribution in the coding regions of the genome of Enterobacteriaceae.
Use of arbitrary primers together with a primer based on Shine-Dalgarno sequences from the 5′-end of bacterial mRNAs.
To decrease the number of false positives caused by rRNA, use antisense probes for 16S and 23S rRNA, which show the fragments amplified from rRNA.

39. Another approach uses a large number of arbitrary primers to allow high-density sampling of differentially expressed genes.
Designed to avoid annealing of the arbitrary primers to stable RNAs such as rRNA.
RAP-PCR has analysed differential gene expression in prokaryotes, in particular for the analysis of genes involved in environmentally relevant processes such as response to pollutants, symbiotic associations and sulfate respiration.

40. Apart from DD and RAP-PCR there is a variety of related techniques relying on reverse transcription and PCR amplification procedures, including cDNA-amplified fragment length polymorphism (cDNA-AFLP), cDNA representational difference analysis (cDNA-RDA), cDNA-RNA subtractive hybridization and most recently DNA microarrays.

41. 5.6 Conclusions
Questions such as which genes are expressed in the environment, how diverse the transcripts are, what are the transcription rates in the environment, how is gene expression affected by changing environmental conditions and which microbes are expressing the genes of interest can be approached by RT-PCR techniques.
Results from RT-PCR studies should significantly contribute to our understanding of the functioning of microbial processes in the environment.
A key challenge is still to obtain sufficient amounts of high quality RNA from the environment, and in particular from environments in which microbial biomass is limited and/or inhibitory substances (such as humic acids) are present.