Venous blood samples were obtained at various time points (Tempus RNA tubes, Thermo Fisher Scientific, Waltham, MA, USA). The first blood sample was drawn on ICU admission (within 24 h). For patients who subsequently developed sepsis and septic shock, 2 more blood samples were obtained within 6 h of their development, respectively. For non-septic patients, a second blood sample was drawn at ICU discharge. The corresponding kit was utilized to isolate total RNA from the peripheral blood, per the manufacturer’s instructions. The concentration and quality of isolated total RNA were measured with a spectrophotometer at 260/280 nm.
Tempus rna tubes
Manufactured by Thermo Fisher Scientific
Sourced in United States
Tempus RNA tubes are designed to stabilize and preserve RNA in whole blood samples. The tubes contain a proprietary reagent that immediately stabilizes RNA upon blood collection, preventing degradation. This allows for reliable RNA analysis from blood samples.
Automatically generated - may contain errors
9 protocols using tempus rna tubes
1
Venous Blood Sampling for RNA Isolation
2
Malaria Parasite DNA and Gametocyte Determination
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Whole blood was drawn by venipuncture at the time of enrollment and collected in EDTA tubes (5 mL) for malaria parasite DNA isolation and qPCR and Tempus RNA tubes (3 mL, Thermofisher) for gametocyte determination. Venous blood samples collected were stored in polystyrene boxes containing frozen gel packs. They were then transported to the nearest health center in under 9 h at a temperature of 5 °C or less. Samples were centrifuged for sera and buffy coat separation. Fractions were frozen at temperatures between −15 and 25 °C and then shipped to Colombia in dry ice. qPCR was performed 1 week after arrival. Samples were handled as potential biohazards and all laboratory staff strictly followed standardized bio-safety procedures.
3
Prospective Study of Health Improvement
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Peripheral blood samples (10 ml) were collected into EDTA tubes that were frozen for DNA preservation, and Tempus RNA tubes (Life Technologies, Grand Island, NY, USA) for preservation of RNA. Samples were taken during regular 6-monthly visits to the Center, generally between the hours of 10:00 a.m. and 12:00 noon. The 74 clinical traits considered here are listed in Additional file 1 : Table S1 and were measured within 2 h of the blood sampling or generated from self-reported survey assessments taken within 1 week of each sample. A total of 668 individuals enrolled and have participated in the CHDWB, which is an ongoing longitudinal study designed to evaluate the impact of health coaches on wellbeing. Across the cohort, significant improvement in most major indicators of health including physical, biochemical, and mental health parameters is observed [10 (link)], and is maintained for at least 3 years, but the effects are modest and tend to be restricted to those individuals with the highest baseline risks.
4
Whole-Blood Gene Expression and SNP Analysis
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We analyzed whole-blood gene expression data from 71 Chinese ethnicity volunteers in the context of their whole-genome SNP profile (data from another ongoing study). mRNA was extracted from whole blood collected into tempus RNA tubes (Life Technologies, Carlsbad, Calif), and transcript abundance was measured by using the Illumina HumanHT-12-v4 Expression Bead Chip (Illumina, San Diego, Calif). The Illumina Human Omni5Quad chip was used to determine the genome-wide SNP profile. Only Illumina probes free of any SNPs were used to determine the expression level of the genes to avoid allelespecific artifacts. Probes used for analysis included the following: ILMN_1657095 (StAR-related lipid transfer [START] domain containing 3 [STARD3]), ILMN_1662174 (ORMDL sphingolipid biosynthesis regulator 3 *Patients with allergic asthma were classified based on a self-reported doctor's diagnosis of asthma with a positive SPT response to one of the allergens tested. Patients with AR were defined by 2 or more self-reported rhinitis symptoms and a positive SPT response. àA total of 337 samples in the discovery cohort and 358 samples in the validation cohort were from patients with both AR and asthma. §Control subjects are healthy subjects classified based on absence of symptoms, a history of allergic disease, and a negative SPT response. There were 1320 total control subjects.
5
Longitudinal study of TB and contacts
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TB patients and household contacts were recruited in a longitudinal study, conducted at Gulab Devi Chest Hospital, Lahore, Pakistan in 2009/2010. Detailed characteristics of subjects, treatment regimen/outcome and recruitment procedures can be found in Table 1 and have been described previously [20 (link)]. The TB patients (n = 26) were recruited prior to starting TB treatment and after two months intensive phase treatment according to the National Tuberculosis Program guidelines (2HRZE). It was only possible to follow up a subset of those patients studied pre-treatment at two months due to early discharge of patients from the hospital. Household contact was defined as living in the same room when the TB patient was diagnosed. QuantiFERON-TB Gold (Cellestis, Ltd, Carnegie, Australia) was used in conjunction with lack of clinical symptoms or radiological evidence of TB to categorise the healthy household contacts as latently infected or not infected with M. tuberculosis.
Three ml venous blood from each patient or contact was collected into Tempus RNA tubes (Applied Biosystems) and stabilised by shaking vigorously for 10 seconds prior to freezing at -80°C.
Three ml venous blood from each patient or contact was collected into Tempus RNA tubes (Applied Biosystems) and stabilised by shaking vigorously for 10 seconds prior to freezing at -80°C.
6
Whole Blood RNA Isolation Protocol
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The total RNA, including miRNAs, was isolated from 3 mL of whole blood that had been collected in Tempus RNA tubes (Applied Biosystems, Life Technologies Corporation, Johannesburg, South Africa) that had been stored at −80 °C. The MagMAX for Stabilized Blood Tubes RNA Isolation Kit was used to perform the extraction, as per the manufacturer’s specifications (Ambion, Life Technologies Corporation, Johannesburg, South Africa). The purity and integrity of the subsequent RNA samples, each with a total volume of 50 μL, was then assessed using a Nanodrop (Nanodrop Technologies, Wilmington, DE, USA), and only samples with an RNA concentration >15 ng/mL, and an OD (optical density) ratio A260/A280 >1.8, were accepted for further processing.
7
Whole Blood RNA Isolation Protocol
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Total RNA, including miRNAs, was isolated from 3ml of whole blood, which was collected in Tempus RNA tubes that were stored at −80°C before RNA isolation (Applied Biosystems). The MagMAX™ for Stabilized Blood Tubes RNA Isolation Kit was used to perform the RNA extraction, as per manufacturer’s specifications (Life Technologies, South Africa). RNA purity and integrity were then evaluated using a nanodrop (nanodrop Technologies, Wilmington, United States), and only samples with a concentration>15ng/ml, and an OD (optical density) ratio A260/A280>1.8, were deemed adequate for further processing.
8
Trauma-Induced Transcriptome Profiling
9
Trauma-Induced Transcriptome Profiling
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RNAseq and questionnaire data were collected from males recruited as part of the Grady Trauma Project, a larger study investigating genetic and environmental risk factors for post-traumatic stress disorder (PTSD) as previously described above. Blood samples were collected following admission to the emergency department after trauma exposure, and questionnaires completed during follow-up assessments at 1, 3, 6, and 12 months post-trauma. Venous blood was collected in Tempus Tubes (RNA, Applied Biosystems) in the ED in the immediate aftermath of trauma exposure. After RNA extraction, mRNA libraries were created using the TrueSeq preparation kit and sequenced on the Illumina HiSeq 2000. Reads were aligned to GRCh37 and the Limma package was used for normalization, converting raw counts to log-CPM, adjusting for library sizes, and reducing heteroscedasticity.
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