Total RNA extraction was performed using the miRNeasy Micro Kit according to the manufacturer’s instructions. Briefly, 500 µl of Qiazol solution (a buffer containing guanidinium thiocyanate, GITC) was used to lyse the tissue. 100 µl of chloroform was added. The mixture was vortexed and centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was obtained, and 1.5 volume of 100% ethanol was added. The mixture was centrifuged through an extraction column (Qiagen S.A.). The column was washed with RWT buffer and REP buffer, respectively. RNA was eluted in 20 µl of RNase-free water and quantified using NanoDrop. RNA samples were stored at –80°C prior to RT-PCR. Complementary DNA (cDNA) was synthesized from total RNA using PrimeScript™ RT Master Mix Kit (Takara). For RT reactions, 100–500 ng of RNA extract was used. The cDNA specimens were stored at −20°C until PCR. Quantitative real-time PCR was performed using SYBR Green with an ABI StepOnePlus system (Life Technologies, Carlsbad, CA, USA). The primers used are listed in Table 2
Extraction column
Manufactured by Qiagen
The Extraction column is a laboratory equipment designed to facilitate the extraction and isolation of specific molecules or compounds from complex samples. It serves as a critical component in various biological and chemical analysis workflows. The core function of the Extraction column is to provide a controlled environment for the selective separation and purification of target analytes from a mixture, enabling researchers to obtain high-purity samples for further analysis and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Mirneasy micro kit, by Qiagen (2 mentions)
Abi steponeplus system, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix kit, by Takara Bio (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Picogreen assay, by Thermo Fisher Scientific (1 mentions)
Ffpe kits, by Qiagen (1 mentions)
5 protocols using extraction column
1
RNA Extraction and qRT-PCR Analysis
2
Urinary Exosome RNA Extraction Protocol
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Total RNA extraction was performed by using miRNeasy Micro Kit according to the manufacturer indications with some modifications (Qiagen S.A.). Briefly, 500 µL Qiazol solution (GITC-containing buffer) was used to dissolve the urinary exosomes. One hundred-microliter Chloroform was added. The mixture was vortexed and centrifuged at 14,000 rpm at 4°C for 15 minutes. The supernatant was obtained and 1.5 volume of 100% ethanol was added. The mixture was centrifuged by the extraction column (Qiagen S.A.). The column was washed respectively by RWT buffer and by REP buffer. The RNA was eluted in 20 µL RNase free water and then quantified by using Nanodrop. The RNA specimens were stored in –80°C until RT-PCR.
3
Exosomal RNA Extraction and Analysis
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We performed the total RNA extraction by using miRNeasy Micro Kit according to the manufacture instructions with some modifications (Qiagen S.A.). Briefly, 500 µl Qiazol solution (GITC-containing buffer) was added to dissolve the exosomes. Then 100 µl chloroform was added. The mixture was vortexed and centrifuged at 14,000 rmp at 4°C for 15 min. The supernatant was secured and 1.5 volume of 100% ethanol was added. The extraction column (Qiagen S.A.) was used to extract the total RNA from the mixture. The column was washed, respectively by RWT buffer and by REP buffer. Finally, the toal RNA was eluted in 17 µl RNase free water. The RNA was then quantified by using Nanodrop. The size distribution of exosomal RNA was analyzed by using a 2100 Bioanalyzer (Agilent Technologies). The RNA specimens were kept in –80°C until use.
4
MGRB and ASRB Participant DNA Processing
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For ASPREE participants of the MGRB, peripheral blood samples were processed to buffy coat within 4 h of collection, then stored at −80 °C. DNA was later purified from buffy coat via magnetic bead extraction (Qiagen).
For 45 and Up Study participants of the MGRB, peripheral blood samples were refrigerated at 4 °C and processed to buffy coat within 48 h of collection. Buffy coat was stored at −80 °C, and DNA purified via column extraction (Qiagen).
ASRB participant PBMCs were extracted from whole blood by centrifugation in Lymphoprep (Vital Diagnostics). Genomic DNA (gDNA) was extracted from PBMCs using salt extraction and quantified by PicoGreen assay (Life Technologies). The integrity of gDNA was determined by agarose gel electrophoresis prior to sequencing.
For 45 and Up Study participants of the MGRB, peripheral blood samples were refrigerated at 4 °C and processed to buffy coat within 48 h of collection. Buffy coat was stored at −80 °C, and DNA purified via column extraction (Qiagen).
ASRB participant PBMCs were extracted from whole blood by centrifugation in Lymphoprep (Vital Diagnostics). Genomic DNA (gDNA) was extracted from PBMCs using salt extraction and quantified by PicoGreen assay (Life Technologies). The integrity of gDNA was determined by agarose gel electrophoresis prior to sequencing.
5
Metastatic Tumor DNA and RNA Isolation
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DNA and RNA isolation from the freshly frozen cryostat-microdissected metastases (n = 9; Fig. 3B) was performed with phenol–chloroform extraction or QIAGEN column extraction. A noncancerous kidney sample was used as germline reference control. Cancer cell fraction (CCF) estimated by visual analysis of hematoxylin and eosin–stained dissected tumor sections and from examination of allelic fractions was between 75% and 90% (mean CCF was 84.6%) for all tumor samples. WGS was performed for all nine metastatic samples and RNA-seq was performed in six of the same metastatic tissue materials (alternating sections used for DNA or RNA isolation). Laser microdissection of selected foci (Fig. 1 B) from the prostate removed 5 yr before death was performed using Arcturus and MMI dissection systems. DNA isolation was performed using QIAGEN FFPE kits.
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