Published: June 11th, 2016
A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.
Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.
Microorganisms are the corner-stone of the biosphere driving ecosystem function. The majority of microorganisms remain uncultured1. Therefore molecular based approaches are fundamental to advance our understanding of the diversity and function of microorganisms in the environment. Central to these approaches is the extraction of nucleic acids from environmental samples and the subsequent amplification of target genes using the Polymerase Chain Reaction (PCR).
The first step of DNA/RNA extraction aims to lyse the cell walls of the microbial community present, remove undesired non nucleic-acid molecules (e.g., organic and inorganic substances) and retain DNA/RNA in solution for further downstream analysis. Among several options available in the literature2,3,4,5, including a range of commercial extraction kits, the Griffiths method6 is widely employed7,8,9. It is cost-effective and particularly well suited to sediments as it uses a bead-beating step to lyse cells and incorporates steps to minimize the co-extraction of PCR inhibitors, such as humic acids, whilst simultaneously recovering DNA and RNA.
The second step uses the polymerase chain reaction (PCR) to amplify target genes, such as the 16S rRNA taxonomic marker, from the extracted nucleic acids. This approach has and continues to facilitate the exploration of the uncharacterized microbial black box10,11. However, end-point PCR-based methods suffer from various limitations that can bias the characterization of microbial communities12. To accurately quantify gene/transcript abundances, real-time PCR, also known as quantitative PCR (qPCR) must be used. qPCR exploits fluorescent reporter dye systems that track amplicon accumulation after every cycle of the PCR. This is significant as it means that quantification can occur during the early exponential phase, rather than the end-point, phase of the PCR reaction, when the amplicon yield is still directly proportional to the initial abundance of the target gene.
Two reporter systems are commonly used: an intercalating nucleic acid stain13 and the 5' 3' exonuclease activity of the DNA polymerase14. Since the former reporter system binds indistinctively to all double-stranded DNA, it may lead to an overestimation of the target sequence if unwanted non-specific amplicons or primer dimers by products occur. In order to circumvent this, extensive optimization of amplification may be required. In the latter system, template amplification is tracked using a combination of a 5' nuclease activity of Taq polymerase that cleaves a fluorophore from an internal probe. This feature increases the specificity of the assay due to the utilization of a fluorogenic probe that binds only to the complementary target-specific sequence between the primer pair. With both chemistries quantification is achieved by determining the crossing-point (Cp) where the accumulation of PCR amplicons, as measured by an increase in fluorescence, is significantly above background fluorescence.
qPCR has been extensively used in microbial ecology to determine gene abundances in different environments15. Moreover, reverse transcription of RNA to cDNA is combined with qPCR and RT-qPCR to quantify gene expression. Hence, qPCR and RT-qPCR represent fast, effective methods for the quantification of gene and/or transcript numbers within environmental samples.
Microorganisms in coastal sediments drive various ecosystem processes, including the mineralization of organic matter, the degradation of pollutants and the biogeochemical cycling of macronutrients such as nitrogen16,17,18. The exhaustive understanding of these transformations requires a comprehensive account of the contributing microbial populations, including quantitative data on gene and transcript abundances. Here we introduce a series of thoroughly tested, streamlined and standardized protocols for the quantification of bacterial 16S rRNA gene and transcript abundances in coastal sediments. The protocol outlines sample collection, simultaneous DNA and RNA extraction, DNA-free RNA preparation, quality checking of extracted nucleic acids, generation of 16S rRNA-DNA and -RNA standards and quantification of environmental samples. Quantitative data derived from the methods described here are needed to shed light on microbial communities driving coastal ecosystems.
1. DNA & RNA Extraction from Marine Coastal Sediments
2. Preparation of RNA and Quality Check of DNA-free RNA
3. Generation of First Strand cDNA from RNA
4. Quantitative PCR
The extraction of good quality DNA and RNA from sediments is the first step in quantifying gene and transcript abundances. A successful extraction yields clear DNA and RNA bands as indicated in Figure 1 for sample A-C, where sharp 23S and 16S rRNA bands are visible in addition to the high molecular weight genomic DNA band.
Figure 1. DNA/RNA extraction. Typical results from DNA/RNA extraction from triplicate (A-B-C) 0.5 g coastal sediments. 5 µl of DNA/RNA was run on a 1.4% agarose at 85 V for 40 min. A molecular marker in the 10,037-200 bp range was used. Please click here to view a larger version of this figure.
To prepare RNA, a digestion of the co-extracted DNA is mandatory. This must be followed by 16S rRNA end-point PCR of the RNA to ensure that the DNA has been successfully removed. If the DNA has been completely removed only a band in the positive control is observed. It is important to use both neat and a 1:10 dilution of RNA to ensure inhibitors are not preventing the formation of a PCR product. RNA can now undergo the reverse transcriptase reaction to convert it to cDNA. This can be performed with either gene-specific primers or random hexamers. Typically, the reaction is carried out in a PCR machine, to ensure optimal temperature profile. This cDNA is used as template in the subsequent qPCR reaction. DNA and RNA is quantified to determine the template concentration used in each reaction.
A qPCR reaction protocol is outlined targeting 16S rRNA DNA. For 16S rRNA transcripts substitute the standard curve and template with cDNA. As unknown samples are quantified against the standard curve, it is imperative to ensure that the standard curve is of good quality. Figure 2 shows the preparation of (A), standard curves and environmental DNA dilutions, (B), amplification of the standard curve and environmental samples (C) conversion of standard curve to a linear regression and calculation of gene copy numbers. When a 10-fold dilution range is correctly prepared and amplified a 3.3 cycle difference between each standard dilution is seen (it takes 3.3 cycles for a ten-fold increase in template at 100% amplification efficiency) (Figure 2Bi). Standard curves descriptors, including the R2 values of 0.99 and PCR efficiencies within the range of 90 to 110% (Figure 2C) are desired. It is important to report Cp values from the no template control (NTC) if present. If this occurs a Cp cutoff for standards and unknown samples 3.3 cycles (i.e., a log fold) higher than the Cp value of the NTC is imposed20 (Figure 2Cii). Unknown template extracted from sediment DNA was quantified from neat, 10-1, 10-2 and 10-3 dilutions (B). The neat sample did not amplify, the Cp values of 10-1 to 10-3 dilutions were 24.12, 26.02 and 28.40 respectively. The NTC Cp was 30.5, an NCT cutoff of 27.2 was imposed. Converting gene abundances to g-1 wet weight sediment resulted in 2.5 x 107 and 7.1 x 107 for 10-1 and 10-2 respectively for the dilution range. 10-3 dilution Cp was below the NTC cutoff, and was therefore not used. In this case the 10-2 was selected as the optimal template dilution.
Figure 2. 16S rRNA gene qPCR amplification of standards and environmental DNA extracted from coastal sediments. Preparation of (A) i) DNA standard curve and ii) environmental DNA dilutions for qPCR amplification, (B) qPCR amplification of i) DNA standard curve and ii) environmental samples, Cp for each sample are shown. (C) i) Linear regression of standard curve with standard curve descriptors; ii) calculation of gene abundances from environmental samples. NTC: Negative Template Control. Please click here to view a larger version of this figure.
|up to 100 ml
Table 1. Preparation of CTAB-phosphate buffer.
|up to 100 ml
|Note: add PEG 6000 slowly under moderate heating and stirring of 50 ml of dH2O.
Table 2. Preparation of PEG-NaCl precipitation solution.
|Undiluted RNA / 1:10 diluted RNA
|10x PCR Buffer
|dNTPs 10 mM
|63F forward primer
|CAG GCC TAA CAC ATG CAA GTC
|518R reverse primer
|ATT ACC GCG GCT GCT GG
Table 3. DNA-free RNA quality check PCR master mix.
|dNTPs 10 mM
|Reverse primer 10 μM/ Random Hexamers 50 µM
|Up to 13 µl
Table 4. cDNA synthesis reaction mix A.
Table 5. cDNA synthesis reaction mix B.
|Annealing T (°C)
|16S rRNA bacteria
|CGG TGA ATA CGT TCY CGG
|Suzuki et al. 2000 19
|GGW TAC CTT GTT ACG ACT T
|CTT GTA CAC ACC GCC CGT C
Table 6. Primer and probes for 16S rRNA q-PCR assay.
|2x Master Mix
|Forward primer (10 μM)
|Reverse primer (10 μM)
|Probe (10 μM)
Table 7. Real time PCR reaction mix.
|Correlation coefficient (R2)
|A measure of the linearity of the standard curve. Ideally it should be R2=1. Value of R2=0.98-0.99 are acceptable.
|A measure of reaction efficiency. Ideally it should be equivalent to -3.32. Value in the range between -3.58 and -3.1.
|If it is 100%, the templates doubles after each thermal cycle during exponential amplification. A good efficiency range is between 90 and 110%.
|Y Intercept (β)
|The theoretical limit of detection of the reaction. Although not used to quantify reaction sensitivity, it is important when comparing different standard curves of the same target.
Table 8. qPCR reaction descriptors.
The combination of DNA/RNA extraction with qPCR provides a fast, accurate, relatively cost-effective method for the sensitive quantification of gene and transcript abundances from a range of environmental samples, such as coastal sediments.
The initial nucleic acid extraction is the critical step to ensure a representative view of the microbial community present is achieved22. A number of limitations need to be considered for the extraction protocol: a) the achievement of total cell lysis, b) co-extraction of inhibitory compounds (e.g., humic acids or polyphenolics), c) contaminating DNA in the RNA fraction, d) rapid degradation of extracted nucleic acids when stored. Precautions must be taken in order to circumvent these limitations. For example, particular care has to be taken to ensure that the extraction of nucleic acids is optimized for the sample type (e.g., sediments, soil, wastewater, etc.). Significant improvements in nucleic acid yield and quality for both DNA and RNA can be achieved by carrying out preliminary experiments23. To minimize the effect of inhibitory compounds on PCR-based applications24, test a range of dilutions from the extracted DNA and RNA. To circumvent the rapid degradation of the extracted DNA/RNA from multiple freeze-thaw cycles and avoid possible loss of genetic information, store multiple small volume aliquots at -80 °C.
When carefully designed, qPCR is a robust, highly reproducible and sensitive method. Notably, the methods for amplification and standard curve construction outlined in this protocol can be adapted for any gene target of interest, including other phylogenetic markers (i.e., archaeal 16S rRNA, fungal 18S rRNA) or genes involved in important functions in the environment. Known limitations in the use of qPCR are: a) the generation of reproducible high-quality standard curve for absolute quantification, b) the choice of primer/probes and optimization of q-PCR assay conditions, c) the use of low quality/sheared nucleic acids, d) the choice of the working dilution of DNA/RNA to avoid inhibition. Moreover, it has to be considered that qPCR technique provides gene/transcript abundances which may not equate to cell counts: this is particularly the case when 16S and 18S rRNA genes are targeted, as microorganisms have different copy numbers of the ribosomal gene in their genome25.
Poor quality standard curves will result in inaccurate quantification of the gene of interest. It is good practice to store a stock of high concentration standards in small aliquots from which fresh standard curves can be made. Do not store standard curves, always make fresh dilutions from a stock of the highest concentration each time. For accurate quantification of environmental samples ensure the range of concentrations of the standard curve spans the expected Cp values of the unknown samples. When quantifying transcripts, construct the standard curve from RNA not double stranded DNA. When possible, quantification of samples to be directly compared should be completed within a single assay to avoid inter-assay variation20. This may not always be possible. Therefore, to compare gene abundances generated between assays it is advisable to have a random sub-set of samples replicated between assays. A wide selection of primer and probe sets are currently available for qPCR targeting microbial taxa and functional groups15. Careful consideration is needed when selecting these to ensure both maximal coverage and specificity for the target group. If the reaction efficiency of a qPCR assay is not satisfactory, troubleshooting starts with testing different thermal cycling conditions (as annealing time and/or temperature) and/or reaction conditions, e.g., varying primer-probe concentrations. Once the q-PCR assay and reaction conditions are optimized, always conduct an initial test of a range of DNA/cDNA dilutions to determine the appropriate template concentration. Select the dilution range resulting in the highest copy number as the optimal template dilution for further assays.
Currently, next-generation sequencing technologies efficiently shed light on microbial community structure and functions in a plethora of environments26,27,28. However, these datasets are often based on end-point PCR amplicon libraries and therefore provide only semi-quantitative assessments of the abundance of particular taxa. Hence, the capability of real time PCR-based technique to target specific taxonomic markers (from higher domain down to strain level) allows efficient validation of the results obtained by next-generation sequencing. Moreover, qPCR has been successfully used in combination with other microbial ecology molecular methods such as stable isotope probing (SIP) or phylogenetic/functional microarrays. Combined with the former tool, qPCR can be used to quantify the metabolically active community29,30. When combined with microarray analysis, qPCR provides key quantitative interpretation of phylogenetic marker-based and functional gene surveys of environments31,32.
Therefore, whether used alone or in combination with other (often process-based) assessments of ecosystem function, quantitative PCR is an essential tool for microbial ecologists in the exploration of the elusive link between microbial communities and ecosystem functions.
The authors have nothing to disclose.
This publication has emanated from research conducted with the financial support of the Natural Environment Research Council (NERC) under grant number NERC NE/JO11959/1 and Science Foundation Ireland & the Marie-Curie Action COFUND under Grant Number 11/SIRG/B2159awarded to CJS and the Eastern Academic Research Consortium (Eastern ARC).
|Toxic, open under chemical hood
|cetrimonium bromide (CTAB)
|Irritant, open under chemical hood
|potassium phosphate dibasic
|potassium phosphate monobasic
|Phenol:Chloroform:Isoamylalcohol pH 8
|Equilibrate at pH 8 before using
|Ethanol Molecular Grade
|Lysing Matrix E tubes
|RNAse/DNAse free 0.2 ml PCR tubes
|pGEM Easy T Vector
|E. coli JM109 competent cells
|Plasmid Midi Kit
|Quant-IT DNA HS Assay
|Quant-IT RNA HS Assay
|TaqMan SensiFast Probe Lo-ROX kit
Copyright © 2024 MyJoVE Corporation. All rights reserved