Name: digital pcr for quantifying circulating micrornas in acute myocardial infarction and cardiovascular disease
Uploaded: 2018-07-03T14:45:00
Description: Watch this Scientific Journal Video about Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease at JoVE.com

The authors have no acknowledgments.

1. Extraction of miRNA from Plasma/Serum
Note: In order to quantify miRNAs appropriately, the correct microRNA isolation from the plasma/serum is a crucial step. A key thing to keep in mind, especially because different protocols exist, is to adhere to the same workflow while processing the samples. In this protocol, miRNA is extracted from 50 &#181;L of serum. Do not use more than 200 &#181;L as this limit the correct extraction process.
<ol>
	<li>Prepare serum or thaw frozen samples.</li>
...

<ol>
	<li>Schulte, C., Zeller, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA-based+diagnostics+and+therapy+in+cardiovascular+disease+-+summing+up+the+facts.">microRNA-based diagnostics and therapy in cardiovascular disease - summing up the facts.</a> Cardiovascular Diagnosis and Therapy. 5, 17-36 (2015).</li><li>Sun, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microRNAs+in+myocardial+infarction:+from+molecular+mechanism+to+clinical+application.">The role of microRNAs in myocardial infarction: from molecular mechanism to clinical application.</a> International Journal of Molecular Sciences. 18, (2017).</li><li>Dimmeler, S., Zeiher, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs:+novel+biomarkers+for+cardiovascular+diseases.">Circulating microRNAs: novel biomarkers for cardiovascular diseases.</a> European Heart Journal. 31, 2705-2707 (2010).</li><li>Creemers, E. E., Tijsen, A. J., Pinto, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs:+novel+biomarkers+and+extracellular+communicators+in+cardiovascular+disease.">Circulating microRNAs: novel biomarkers and extracellular communicators in cardiovascular disease.</a> Circulation Research. 110, 483-495 (2012).</li><li>Oerlemans, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+assessment+of+acute+coronary+syndromes+in+the+emergency+department:+the+potential+diagnostic+value+of+circulating+microRNAs.">Early assessment of acute coronary syndromes in the emergency department: the potential diagnostic value of circulating microRNAs.</a> EMBO Molecular Medicine. 4, 1176-1185 (2012).</li><li>Olivieri, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+potential+of+circulating+miR-499-5p+in+elderly+patients+with+acute+non+ST-elevation+myocardial+infarction.">Diagnostic potential of circulating miR-499-5p in elderly patients with acute non ST-elevation myocardial infarction.</a> Internation Journal of Cardiology. 167, 531-536 (2013).</li><li>Navickas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+circulating+microRNAs+as+biomarkers+of+cardiovascular+disease:+a+systematic+review.">Identifying circulating microRNAs as biomarkers of cardiovascular disease: a systematic review.</a> Cardiovascular Research. 111, 322-337 (2016).</li><li>Devaux, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+and+prognostic+value+of+circulating+microRNAs+in+patients+with+acute+chest+pain.">Diagnostic and prognostic value of circulating microRNAs in patients with acute chest pain.</a> Journal of Internal Medicine. 277, 260-271 (2015).</li><li>Schwarzenbach, H., da Silva, A. M., Calin, G., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+normalization+strategies+for+microRNA+quantification.">Data normalization strategies for microRNA quantification.</a> Clinical Chemistry. 61, 1333-1342 (2015).</li><li>Hindson, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+droplet+digital+PCR+system+for+absolute+quantitation+of+DNA+copy+number.">High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.</a> Analytical Chemistry. 83, 8604-8610 (2011).</li><li>Hindson, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+quantification+by+droplet+digital+PCR+versus+analog+real-time+PCR.">Absolute quantification by droplet digital PCR versus analog real-time PCR.</a> Nature Methods. 10, 1003-1005 (2013).</li><li>Robinson, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+digital+PCR+as+a+novel+detection+method+for+quantifying+microRNAs+in+acute+myocardial+infarction.">Droplet digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction.</a> Internation Journal of Cardiology. 257, 247-254 (2018).</li><li>Mitchell, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+as+stable+blood-based+markers+for+cancer+detection.">Circulating microRNAs as stable blood-based markers for cancer detection.</a> Proceedings of the National Academy of Sciences of the United States of America. , 10513-10518 (2008).</li><li>D'Alessandra, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+are+new+and+sensitive+biomarkers+of+myocardial+infarction.">Circulating microRNAs are new and sensitive biomarkers of myocardial infarction.</a> European Heart Journal. 31, 2765-2773 (2010).</li><li>Forootan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+determine+limit+of+detection+and+limit+of+quantification+in+quantitative+real-time+PCR+(qPCR).">Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR).</a> Biomolecular Detection and Quantification. 12, 1-6 (2017).</li><li>Dingle, T. C., Sedlak, R. H., Cook, L., Jerome, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+of+droplet-digital+PCR+vs+real-time+quantitative+PCR+to+inhibitory+substances.">Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances.</a> Clinical Chemistry. 59, 1670-1672 (2013).</li><li>Taylor, S. C., Laperriere, G., Germain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+digital+PCR+versus+qPCR+for+gene+expression+analysis+with+low+abundant+targets:+from+variable+nonsense+to+publication+quality+data.">Droplet digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data.</a> Scientific Reports. 7, 2409 (2017).</li></ol>

Digital PCR is a relatively novel end-point method of PCR that allows the direct absolute quantification of nucleic acids within a sample. The method possesses particular advantages, including a decreased variability, an increased day-to-day reproducibility, and a superior sensitivity11,12. Further, due to the partitioning of the sample into approximately 20,000 single reactions and endpoint analyses, dPCR is more robust to interfering substances in PCR compared ...

Circulating miRNAs have been identified as promising markers for a number of diseases, including cardiovascular disease1. The miRNAs are small, non-coding single-stranded RNA molecules (approximately 22 nucleotides long) are involved in the post-transcriptional regulation via the alteration of the messenger RNA translation and influencing gene expression2, and are released into the circulation in both physiological and pathological states. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity1. In cardiovascular diseases, miRNAs have become attractive candidates as novel biomarkers because they are remarkably stable in the serum and can easily be quantified with the help of PCR methodology3. The potential value of miRNAs as biomarkers for myocardial infarction has been evaluated in small studies, but a validation in large cohorts is lacking2. For example, miR-499 is found highly expressed in the myocardial muscle, and it has been shown to be significantly increased in an AMI4,5,6. Further, it regulates programmed cell death (apoptosis) and the differentiation of cardiomyocytes and is thus involved in several mechanisms following an AMI7. Apart from some small studies reporting a superiority and incremental value of miRNAs for the diagnosis of AMI, the superiority or equality to high-sensitivity cardiac troponins has not yet been proven in large-scale studies2,5,6,8. More prospective studies in large cohorts are, therefore, needed to assess the potential diagnostic value of miRNAs. Additionally, methods of miRNA quantification need to be optimized and standardized using comparable protocols9. Standardized assays may reduce inconsistent results and may help miRNAs to become potential biomarkers for the routine clinical application, as biomarkers need to be quantified in a reproducible way to ensure their clinical applicability.
Recently, dPCR has been introduced as an end-point analysis. It partitions the sample into approximately 20,000 individual reactions10. The dPCR system then utilizes a mathematical Poisson statistical analysis of fluorescent signals (positive and negative reactions), enabling an absolute quantification without a standard curve10. When combining dPCR with fluorescent hydrolysis probes, the highly specific direct absolute quantification of miRNAs is made possible. Digital polymerase chain reaction has shown to exhibit superior technical qualities (including a decreased variability, an increased day-to-day reproducibility, a high degree of linearity, and a high sensitivity) for quantifying miRNA levels in the circulation compared to quantitative real-time PCR10,11. These superior technical qualities might help to mitigate current limitations on using circulating miRNAs as biomarkers and may lead to the establishment of miRNAs as biomarkers in large multi-center cardiovascular clinical trials and as a diagnostic method in general. In a previous study, we recently applied dPCR for the absolute quantification of circulating miRNAs in patients with an AMI and were able to demonstrate superior diagnostic potential compared to the quantification of miRNAs by qPCR12.
In this publication, we want to demonstrate that using dPCR is an accurate and reproducible method for directly quantifying circulating cardiovascular miRNAs. The absolute quantification of miRNA levels in serum, using digital PCR, shows potential for the use in large multi-center cardiovascular clinical trials. In this publication, we describe in detail how to effectively perform digital PCR and how to detect the absolute miRNA copy number in serum.

<table>
	<tbody>
		<tr>
			<td>RNA-Extraction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Kit (50)</td>
			<td>Qiagen-Sample &#38; Assay Technologies, Hilden, Deutschland</td>
			<td>217184</td>
			<td>Kit for microRNA extraction. Kit contains commercial buffer RWT (called number one in the manuscript)&#160; and RPE (called number two in manuscript).</td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Spike-in-Control; Syn-cel-miR-39 miRNA; 10pmol</td>
			<td>Qiagen-Sample &#38; Assay Technologies, Hilden, Deutschland</td>
			<td>219610</td>
			<td>Spike-in for normalisation , Sequence: 5&#39;-UCACCGGGUGUAAAUCAGCUUG-3&#39;</td>
		</tr>
		<tr>
			<td>Reverse Transcription</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit (1000 Reactions)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4366597</td>
			<td>Kit for microRNA reverse transcription</td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Assays M</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assays used in reverse transcription</td>
		</tr>
		<tr>
			<td>hsa-miR-499 (750 RT/750 PCR rxns)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assay Number 001352</td>
		</tr>
		<tr>
			<td>cel-miR-39 (750 RT/750 PCR rxns)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assay Number 000200</td>
		</tr>
		<tr>
			<td>PCR Plate, 96-well, segmented, semi-skirted</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>AB0900</td>
			<td>96 well plate for reverse transcription</td>
		</tr>
		<tr>
			<td>Microseal &#8216;B&#8217; seal Seals</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>MSB1001</td>
			<td>Foil to ensure proper storage</td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1851196</td>
			<td>Cycler used for reverse transcription</td>
		</tr>
		<tr>
			<td>Droplet Digital PCR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 nmole RNA oligo hsa-miR-499-5p</td>
			<td>Integrated DNA Technologies</td>
			<td>Custom</td>
			<td>Sequence: 5&#39;-phos-UUAAGACUUGCAGUGAUGUUU-3&#39;</td>
		</tr>
		<tr>
			<td>ddPCR Supermix for Probes (No dUTP)</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863024</td>
			<td>Supermix used in droplet generation</td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Assays</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assays used in digital PCR (fluorescent hydrolysis probe)</td>
		</tr>
		<tr>
			<td>hsa-miR-499 (750 RT/750 PCR rxns)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assay Number 001352, commercial primers</td>
		</tr>
		<tr>
			<td>cel-miR-39 (750 RT/750 PCR rxns)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assay Number 000200, commercial primers</td>
		</tr>
		<tr>
			<td>DG8 Cartridges and Gaskets</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1864007</td>
			<td>Cartridge takes up to 8 samples for droplet generation</td>
		</tr>
		<tr>
			<td>DG8 Cartridge Holder</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863051</td>
			<td>Holds cartridges in droplet generation</td>
		</tr>
		<tr>
			<td>Droplet Generation Oil for Probes</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863005</td>
			<td>Oil used in droplet generation</td>
		</tr>
		<tr>
			<td>ddPCR 96-Well Plates</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>12001925</td>
			<td>96 well plate for ddPCR</td>
		</tr>
		<tr>
			<td>PCR Plate Heat Seal, foil, pierceable</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1814040</td>
			<td>Pierceable foil, compatible with droplet reader</td>
		</tr>
		<tr>
			<td>ddPCR Droplet Reader Oil</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863004</td>
			<td>Oil used in droplet reading</td>
		</tr>
		<tr>
			<td>QX100 or QX200 Droplet Generator</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863002</td>
			<td>Droplet Generator, generates the droplets from sample/oil emulsion</td>
		</tr>
		<tr>
			<td>PX1 PCR Plate Sealer</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1814000</td>
			<td>Seals the plate before PCR</td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1851196</td>
			<td>Cycler used for ddPCR</td>
		</tr>
		<tr>
			<td>QX100 or QX200 Droplet Reader</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863003</td>
			<td>Reads PCR-positive and PCR-negative droplets with an optical detector</td>
		</tr>
		<tr>
			<td>ddPCR Buffer Control for Probes</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863052</td>
			<td>Blank control&#160;and to fill up the remaining wells of 8-well cassette</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuantaSof Software</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1864011</td>
			<td>Program for droplet reading</td>
		</tr>
		<tr>
			<td>Prism Windows 5</td>
			<td>GraphPad Software Inc., La Jolla, CA, USA</td>
			<td></td>
			<td>Program for statistical analysis</td>
		</tr>
	</tbody>
</table>

digital pcr for quantifying circulating micrornas in acute myocardial infarction and cardiovascular disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Digital PCR combined with fluorescent hydrolysis probes enables researchers to directly quantify the absolute amounts of specific miRNAs in copies/&#181;L. As the sample in dPCR is partitioned in approximately 20,000 individual PCR reactions, dPCR does not require technical replicates10. The dPCR system utilizes a mathematical Poisson statistical analysis of fluorescent signals (differing between positive and negative reactions) enabling an absolute quantification ...

Watch this Scientific Journal Video about Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease at JoVE.com

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

This method may help answer key questions in the diagnostic field, such as whether micro RNAs are valid biomarkers in cardiovascular disease. Compared to quantitative real-time PCR, digital PCR exhibits superior technical qualities for quantifying microRNAs in the circulation. As the sample is divided into about 20, 000 droplets, no duplicate measurements are necessary, and no standard curve is needed for quantification.Although this method may provide insight into quantification of microRNAs in cardiovascular patients, it may also be applied to other patients and diseases, as other microRNAs have shown promise as biomarkers in cancer progression, it may also be applied to oncological patients. Generally, individuals new to this method will struggle because the generated droplets are fragile and need to be handled with care. After isolating RNA from serum samples, continue with reverse transcription to obtain more stable complementary DNA for dPCR.To begin, thaw the reverse transcription kit on ice. Then spin the enzymes and the primers down. Next, prepare the master mix as described in the text protocol.Combine 10 microliters of the master mix with five microliters of extracted RNA in each well of a 96-well plate. Then centrifuge the plate at 2, 000xg at four degrees Celsius for two minutes. Proceed with the reverse transcription as detailed in the text protocol.First, thaw the commercial dPCR mix, cDNA and PCR primers on ice and centrifuge immediately before use. Then prepare the master mix as described in the text protocol. Add 1.33 microliter volumes of reverse transcription product to 18.67 microliters volumes of the master mix and centrifuge briefly.Using a pipet, transfer 20 microliters of the sample into each well of the medium row of an eight well cassette. Then pipet 70 microliters of droplet generation oil into the oil wells of the cassette. Next, place a gasket on top of the cassette and place it in the droplet generator.Close the droplet generator and wait until the three indicator lights are solid green. Using a pipet, carefully transfer 40 microliters of the droplet-formed sample into separate wells of a 96-well plate. Then seal the plate with dPCR foil at 180 degrees Celsius for four seconds.Place the sealed PCR plate in the cycler and follow the cycling instructions outlined in the text protocol. Remove the plate from the thermocycler and transfer it to the base of a plate holder. Then place the top of the plate holder onto the PCR plate.Finally, start the commercial dPCR software. In this protocol, digital PCR was used to quantify microRNAs in the serum of patients with cardiovascular disease. Samples were taken from 16 patients before, eight hours after, and 16 hours after percutaneous intervention.Patients with ST-elevation myocardial infarction showed a significant increase in miR-499 levels compared to patients with stable coronary artery disease. Once mastered, this technique can be done in about an hour for droplet formation of 96 samples if it is done correctly. While attempting this procedure, it is important to keep in mind to handle the droplets with care.After watching this video, you should have a good understanding of how to perform digital PCR correctly, by correct droplet formation, cycling, reading, and analyzing.

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