Quantification of mRNA in single cells and modelling of RT-qPCR induced noise

<p>Abstract</p> <p>Background</p> <p>Gene expression has a strong stochastic element resulting in highly variable mRNA levels between individual cells, even in a seemingly homogeneous cell population. Access to fundamental information about cellular mechanisms, such as correlated gene expression, motivates measurements of multiple genes in individual cells. Quantitative reverse transcription PCR (RT-qPCR) is the most accessible method which provides sufficiently accurate measurements of mRNA in single cells.</p> <p>Results</p> <p>Low concentration of guanidine thiocyanate was used to fully lyse single pancreatic β-cells followed by RT-qPCR without the need for purification. The accuracy of the measurements was determined by a quantitative noise-model of the reverse transcription and PCR. The noise is insignificant for initial copy numbers >100 while at lower copy numbers the noise intrinsic of the PCR increases sharply, eventually obscuring quantitative measurements. Importantly, the model allows us to determine the RT efficiency without using artificial RNA as a standard. The experimental setup was applied on single endocrine cells, where the technical and biological noise levels were determined.</p> <p>Conclusion</p> <p>Noise in single-cell RT-qPCR is insignificant compared to biological cell-to-cell variation in mRNA levels for medium and high abundance transcripts. To minimize the technical noise in single-cell RT-qPCR, the mRNA should be analyzed with a single RT reaction, and a single qPCR reaction per gene.</p

Waist Circumference is not Superior to Body Mass Index in Predicting Groin Hernia Repair in Either Men or Women

Background and aims: A high body mass index (BMI) is considered a risk factor for ventral abdominal wall hernias but protective for the development of groin hernias. The reason for this is unclear. The surrounding abdominal fat in obesity might “protect” and limit the passage through the inguinal canal. The aim was to compare two different methods used for obesity registration in groin hernia patients and to investigate the hypothesis of high BMI/low groin hernia risk phenomenon. Methods: This was a population-based observational study comparing BMI to waist circumference (WC) as well as their correlations to the quantity of groin hernia repair performed in either sex. Two national registers were crosslinked to a large regional register including information on WC. Results: A larger WC and a higher BMI were associated with a lower risk of having groin hernia repair in both sexes. There was no difference using either WC or BMI as a risk factor for groin hernia repair in either sex. There was no advantage to using body composition based on WC rather than BMI for surgery indication. Conclusions: Overweight patients, both men and women, have a lower risk of undergoing groin hernia repair regardless of fat distribution. BMI is a well-established method for obesity registration and is recommended in the evaluation of hernia patients

Tobacco use is not associated with groin hernia repair, a population-based study

Purpose The pathogenesis of groin hernia is not fully understood and some suggested risk factors are debatable. This population-based study evaluates the association between groin hernia repair and tobacco use. Method An observational study based on register linkage between the Swedish Hernia Register and the Vasterbotten Intervention Program (VIP). All primary groin hernia repairs performed from 2001 to 2013 in the county of Vasterbotten, Sweden, were included. Results VIP provided data on the use of tobacco in 102,857 individuals. Neither smoking nor the use of snus, increased the risk for requiring a groin hernia repair. On the contrary, heavy smoking decreased the risk for men, HR 0.75 (95% CI 0.58-0.96), as did having a BMI over 30 kg/m 2 HR (men) 0.33 (95% CI 0.27-0.40). Conclusion Tobacco use is not a risk factor for requiring a groin hernia repair, whereas having a low BMI significantly increases the risk

Quantitative Transcription Factor Analysis of Undifferentiated Single Human Embryonic Stem Cells

BACKGROUND: Human embryonic stem cells (hESCs) require expression of transcription factor genes POU5F1 (POU class 5 homeobox 1), NANOG (Nanog homeobox), and SOX2 [SRY (sex determining region Y)-box 2] to maintain their capacity for self-renewal and pluripotency. Because of the heterogeneous nature of cell populations, it is desirable to study the gene regulation in single cells. Large and potentially important fluctuations in a few cells cannot be detected at the population scale with microarrays or sequencing technologies. We used single-cell gene expression profiling to study cell heterogeneity in hESCs. METHODS: We collected 47 single hESCs from cell line SA121 manually by glass capillaries and 57 single hESCs from cell line HUES3 by flow cytometry. Single hESCs were lysed and reverse-transcribed. Reverse-transcription quantitative real-time PCR was then used to measure the expression POU5F1, NANOG, SOX2, and the inhibitor of DNA binding genes ID1, ID2, and ID3. A quantitative noise model was used to remove measurement noise when pairwise correlations were estimated. RESULTS: The numbers of transcripts per cell varied >100-fold between cells and showed lognormal features. POU5F1 expression positively correlated with ID1 and ID3 expression (P < 0.05) but not with NANOG or SOX2 expression. When we accounted for measurement noise, SOX2 expression was also correlated with ID1, ID2, and NANOG expression (P < 0.05). CONCLUSIONS: We demonstrate an accurate method for transcription profiling of individual hESCs. Cell-to-cell variability is large and is at least partly nonrandom because we observed correlations between core transcription factors. High fluctuations in gene expression may explain why individual cells in a seemingly undifferentiated cell population have different susceptibilities for inductive cues. (C) 2009 American Association for Clinical Chemistr

Management of groin hernia repair in Sweden : a register-based comparative analysis of public and private healthcare providers

Background: Swedish healthcare is in a period of transition with an expanding private sector. This study compares quality of outcome after groin hernia repair performed in a public or private healthcare setting. Methods: A cohort study based on data from the Swedish National Hernia Register combined with Patient-Reported Outcome Measures (PROMs) 1 year after groin hernia repair. Between September 2012 and December 2018, a questionnaire was sent to all patients registered in the hernia register 1 year after surgery. Endpoints were reoperation for recurrence, chronic pain, and patient satisfaction. Results: From a total of 87,650 patients with unilateral groin hernia repair, 61,337 PROM answers (70%) were received from 71 public and 28 private healthcare providers. More females, acute and recurrent cases, and patients with high American Society of Anesthesiology (ASA) scores were operated under the national healthcare system. The private sector had more experience surgeons with higher annual volume per surgeon, shorter time on waiting lists, and shorter operation times. No difference was seen in patient satisfaction. Groin hernia repair performed in a private clinic was associated with less postoperative chronic pain (OR 0.85, 95% CI 0.8–0.91) but a higher recurrence rate (HR 1.41; 95% CI 1.26–1.59) in a multivariable logistic regression analysis. Conclusion: Despite private clinics having a higher proportion of experienced surgeons and fewer complex cases, the recurrence rate was higher, whereas the risk for chronic postoperative pain was higher among patients treated in the public sector

Data measurements for (left), (centre), and (right) with lines showing upper and lower 95% confidence intervals for different amounts of mRNA based on a log-normal distribution with parameters estimated from the experimental data

<p><b>Copyright information:</b></p><p>Taken from "Quantification of mRNA in single cells and modelling of RT-qPCR induced noise"</p><p>http://www.biomedcentral.com/1471-2199/9/63</p><p>BMC Molecular Biology 2008;9():63-63.</p><p>Published online 17 Jul 2008</p><p>PMCID:PMC2483285.</p><p></p

Overlaid curves show the total technical noise levels for RT-qPCR measurements

The biological noise, which can be interpreted as normalized width of each histogram, is 1.0, 0.33 and 1.4 for and respectively.<p><b>Copyright information:</b></p><p>Taken from "Quantification of mRNA in single cells and modelling of RT-qPCR induced noise"</p><p>http://www.biomedcentral.com/1471-2199/9/63</p><p>BMC Molecular Biology 2008;9():63-63.</p><p>Published online 17 Jul 2008</p><p>PMCID:PMC2483285.</p><p></p