|
Status |
Public on Jul 19, 2023 |
Title |
Latent Tuberculosis 6 |
Sample type |
RNA |
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|
Source name |
Latent Tuberculosis
|
Organism |
Homo sapiens |
Characteristics |
tissue: blood age: 29 gender: Female
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA including miRNA was isolated from the sorted monocytes (Range: 1 to 2.8 million cells) in RLT buffer using Phenol-Chloroform and Qiagen miRNAeasy mini kit (Cat#217004). Samples were extracted with standard Phenol-chloroform mixture. Aqueous phase obtained after centrifugation was mixed with 700 µl of QIAzol Lysis reagent and mixed well. Lysate was incubated at room temperature for 5 minutes and 140 µl of chloroform was added for phase separation. Rest of the protocol was followed as per manufacturer’s guidelines. RNA was eluted in 20µl of nuclease free water (Ambion, Cat # AM9932). Quantitation was performed using Qubit RNA HS assay (Invitrogen, Cat # Q32855) kit and also qualitatively analysed on Agilent 2100 bioanalyzer nano chip (Agilent, Cat # 5067-1511). 100ng of total RNA was used for miRNA assay.
|
Label |
not provided
|
Label protocol |
NanoString uses combination of oligonucleotide based barcodes. Each probe hybridising the miRNAs will be unique with combination of color dye combination which are barcoded barcodes.
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|
|
Hybridization protocol |
Total RNA was processed using the Nanostring nCounter v3.0a array.
|
Scan protocol |
Hybridized molecules are loaded to Nanostring sprint catridge and loaded onto nCounter SPRINT machine for scanning the barcodes. Raw counts data (.RCC) was retrieved from the machine after the scanning completes.
|
Data processing |
Raw data were normalized using nSolver 4.0 software. Normalization of the raw data was performed using the geometric mean of positive controls and top 100 highly expressed miRNAs (CV% < 50) as per the instructions in the manual (nCounter Data Analysis Guidelines for miRNA (LBL-C0046-01) and nSolverTM 4.0 Analysis Software User Manual (MAN-C0019-08)). NSolver 4.0 software was used to compute Normalized counts.
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|
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Submission date |
Apr 05, 2023 |
Last update date |
Jul 19, 2023 |
Contact name |
Ramalingam Bethunaickan |
E-mail(s) |
bramalingam@gmail.com
|
Phone |
9677074680
|
Organization name |
ICMR-National Institute for Research in Tuberculosis
|
Department |
Immunology
|
Lab |
Molecular Immunology
|
Street address |
No.1. Mayor Satya murthy Road, Chetpet
|
City |
Chennai |
State/province |
Tamil Nadu |
ZIP/Postal code |
600031 |
Country |
India |
|
|
Platform ID |
GPL24158 |
Series (1) |
GSE229020 |
Down regulation of Monocyte miRNAs: Implications for Immune Dysfunction and Disease Severity in Drug-Resistant Tuberculosis |
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