RNA was isolated from thalamic tissue punches obtained from five randomly selected animals per group in experiment #3 (see Table 1 ). RNA extraction was performed using the RNA Lipid Tissue Kit from Qiagen (Valencia, CA). The RNA concentration in the resulting sample was determined on a Nanodrop2000. A one-step reverse transcription PCR reaction was performed on BioRAD C1000 Thermal Cycler using the SYBR-Green 1-Step RT-PCR kit and primers from Qiagen (Table 2 ). The thermal protocol was 30 min @ 50 °C for the reverse transcription reaction, 15 min @ 95 °C for DNA pol activation and 40x (15 s @ 94 °C melting, 30 s @ 56 °C annealing, 30 s @ 72 °C extension). A melting curve was obtained thereafter for quality assurance. Sample amount was adjusted according to total RNA concentration to obtain 20 ng of total RNA per well in the final reaction mix. All reactions were run in triplicate. PCR runs that did not exhibit a proper amplification profile were discarded. For each sample, the threshold Ct value for GAPDH was subtracted from the Ct of value for the genes in Table 2 to give a ΔCt. The mean ΔCt from the VZV/high E2 group was subtracted from the VZV/high E2 + ICI 182,780 group to give a ΔΔCt. To get the fold change the −ΔΔCt was raised to the second power (2−ΔΔCt).
Rna lipid tissue kit
Manufactured by Qiagen
Sourced in United States
The RNA Lipid Tissue Kit is a product designed for the isolation and purification of total RNA from lipid-rich tissue samples. It is a tool for researchers and scientists working with RNA extraction and analysis.
Automatically generated - may contain errors
5 protocols using rna lipid tissue kit
1
Thalamic RNA Extraction and qPCR Analysis
2
RNA-Seq of Brain Tissue Samples
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RNA isolation was performed on 22 unaffected control, 25 NA‐GBM, and 11 AD brain tissue samples. A total of 35 brain tissue samples from five unaffected control individuals, five NA‐GBM, and five AD patient donors were selected for RNA‐sequencing (see Section 2.3 ). Approximately 40 mg of frozen brain tissue was processed using Qiagen RNA Lipid Tissue Kit. Quality of the RNA was determined using TapeStation, only samples with a RIN >4.5 were included in the experiment. 70 ng of sample RNA was used for library preparation with the Lexogen QuantSeq 3′ mRNA‐Seq Library Prep Kit (FWD). cDNA libraries were pooled equimolarly and approximately 5 M reads per sample were sequenced on a NextSeq 500 at the sequencing facility in the UMCG.
3
Thalamic RNA Extraction and qPCR Analysis
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RNA was isolated from thalamic tissue punches obtained from four randomly selected animals per group in Experiment #2 (see Table 1 ). RNA extraction was performed using the RNA Lipid Tissue Kit from Qiagen (Valencia, CA, USA). The RNA concentration in the resulting sample was determined on a Nanodrop2000. A one-step reverse transcription polymerase chain reaction (PCR) reaction was performed on BioRAD C1000 Thermal Cycler using the SYBR-Green 1-Step RT-PCR kit and primers from Qiagen (Table 2 ). The thermal protocol was 30 min @ 50°C for the reverse transcription reaction, 15 min @ 95°C for DNA pol activation and 40× (15 s @ 94°C melting, 30 s @ 56°C annealing, 30 s @ 72°C extension). A melting curve was obtained thereafter for quality assurance. Sample amount was adjusted according to total RNA concentration to obtain 20 ng of total RNA per well in the final reaction mix. All reactions were run in triplicate. PCR runs that did not exhibit a proper amplification profile were discarded. For each sample, the threshold Ct value for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was subtracted from the Ct of value for VGAT or the genes in Table 2 to give a ΔCt. Values for decreased expression were calculate by using the formula (−1/ΔCt).
4
Cerebellum RNA Extraction and RT-PCR
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Cerebella were collected from 5-week-old animals and subjected to Dounce homogenization. RNA from individual cerebella was isolated after homogenization with a Lipid Tissue RNA Kit (Qiagen) following the manufacturer's instructions. RNA (1 µg) was used to create cDNA with an ImpromII Reverse Transcriptase Kit (Promega) following the manufacturer's standard protocol, including oligodT oligos and 4.8 M MgCl2 in each 20 µl reaction (Promega). Each reaction (1 µl) was used as template in subsequent PCR amplification reactions in a final volume of 50 µl with GoTaq Green Mastermix (Promega) in the presence of 0.2 M of each oligo under the following conditions: denaturation at 94°C for 3 min; 30 cycles of 94°C for 30 s; 55°C for 30 s; and 72°C for 30 s; followed by a final extension of 72°C for 7 min. To detect the hRonin-Flag transgene mRNA the oligos MAD041 and MAD224 (see above) were used, whereas Actb mRNA was detected with the oligos MAD233 (5′-GGCCCAGAGCAAGAGAGGTATCC-3′) and MAD234 (5′-ACGCACGATTTCCCTCTCAGC-3′), resulting in PCR fragments of 578 and 466 base pairs, respectively.
5
Cerebellum RNA Isolation and qPCR Protocol
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Cerebella were collected from 5-week-old animals and subjected to dounce homogenization.
RNA from individual cerebella was isolated after homogenization with the Lipid Tissue RNA Kit (Qiagen) following the manufacturer's instructions. 1 µg of RNA was used to create cDNA with the ImpromII Reverse Transcriptase Kit (Promega) following the manufactures standard protocol including oligodT oligos and 4.8 M MgCl2 in each 20 µl reaction (Promega). 1 µl of each reaction was used as template in subsequent PCR amplification reactions in a final volume of 50 µl with the GoTaq Green Mastermix (Promega) in the presence of 0.2 M of each oligo under following conditions: denaturation at 94 ˚C for 3 min, 30 cycles of 94 ˚C for 30 sec, 55 ˚C for 30 sec and 72 ˚C for 30 sec, followed by a final extension of 72 ˚C for 7 min. To detect the hRonin-Flag transgene mRNA the oligos MAD041 and MAD224 (see above) were used, while Actb mRNA was detected with the oligos MAD233 (GGC CCA GAG CAA GAG AGG TAT CC) and MAD234 (ACG CAC GAT TTC CCT CTC AGC) resulting in PCR fragments of 578 and 466 base pairs respectively.
RNA from individual cerebella was isolated after homogenization with the Lipid Tissue RNA Kit (Qiagen) following the manufacturer's instructions. 1 µg of RNA was used to create cDNA with the ImpromII Reverse Transcriptase Kit (Promega) following the manufactures standard protocol including oligodT oligos and 4.8 M MgCl2 in each 20 µl reaction (Promega). 1 µl of each reaction was used as template in subsequent PCR amplification reactions in a final volume of 50 µl with the GoTaq Green Mastermix (Promega) in the presence of 0.2 M of each oligo under following conditions: denaturation at 94 ˚C for 3 min, 30 cycles of 94 ˚C for 30 sec, 55 ˚C for 30 sec and 72 ˚C for 30 sec, followed by a final extension of 72 ˚C for 7 min. To detect the hRonin-Flag transgene mRNA the oligos MAD041 and MAD224 (see above) were used, while Actb mRNA was detected with the oligos MAD233 (GGC CCA GAG CAA GAG AGG TAT CC) and MAD234 (ACG CAC GAT TTC CCT CTC AGC) resulting in PCR fragments of 578 and 466 base pairs respectively.
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