Hematoxylin and eosin stain was performed and the slides were reviewed to ensure that at least 85–90% of cells were cancer cells before specimen collection. Total cellular RNA was isolated from either fresh-frozen xenograft tumor pieces using the TRIzol Plus RNA Purification Kit (Life Technologies) or from formalin-fixed, paraffin-embedded (FFPE) specimens (8 x 12-μM sections) using the miRNeasy FFPE Kit (Qiagen), according to the manufacturer’s protocols. Total RNA was eluted from the columns in nuclease-free water and stored at -80°C. RNA concentration and purity were assessed with a NanoDrop 2000 Spectrophotometer (Thermo Scientific) and quality assessments (e.g., RNA integrity) were made using an Agilent 2100 Bioanalyzer (Agilent Technologies). For miRNA expression profiling, total cellular RNA (including the small RNA fraction) was isolated from FFPE bladder cancer specimens using the miRNeasy FFPE Kit (Qiagen) according to the manufacturer’s protocol.
Mirneasy ffpe kit
Manufactured by Qiagen
Sourced in Germany, United States, Netherlands, Italy, United Kingdom, Australia, China
The MiRNeasy FFPE Kit is a product designed for the purification of total RNA, including small RNAs, from formalin-fixed, paraffin-embedded (FFPE) tissue samples. It is a tool for researchers to extract and isolate RNA from these types of samples.
Automatically generated - may contain errors
Lab products found in correlation
Mirneasy mini kit, by Qiagen (35 mentions)
Trizol reagent, by Thermo Fisher Scientific (27 mentions)
2100 bioanalyzer, by Agilent Technologies (24 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (24 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (24 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (21 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (20 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (19 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (19 mentions)
Deparaffinization solution, by Qiagen (18 mentions)
Miscript 2 rt kit, by Qiagen (18 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (16 mentions)
Mirneasy kit, by Qiagen (15 mentions)
Rneasy mini kit, by Qiagen (13 mentions)
Trizol, by Thermo Fisher Scientific (13 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (12 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (12 mentions)
Miscript sybr green pcr kit, by Qiagen (11 mentions)
Mirneasy serum plasma kit, by Qiagen (11 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (11 mentions)
453 protocols using mirneasy ffpe kit
1
RNA Extraction from Tumor Samples
2
Microdissection and RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After microdissection, the cut regions were placed into a collection vessel and underwent a deparaffinization step. The deparaffinization technique used the melting protocol due to the small quantity of the samples. Paraffin was melted and cooled, and the solid layer was pierced with a tip, as indicated by the manufacturer for the miRNeasy FFPE kit (Qiagen, Hilden, Germany).
Total RNA was extracted using the same miRNeasy FFPE kit (Qiagen, Germany) according to the manufacturer’s instructions, using the spin column technique for RNA purification, which included ethanol precipitation of nucleic acids.
Following extraction, we eluted the samples with 12 μL RNase-free water and quantified the RNA concentration with the Qubit RNA HS Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) using a Qubit® Fluorometer.
Total RNA was extracted using the same miRNeasy FFPE kit (Qiagen, Germany) according to the manufacturer’s instructions, using the spin column technique for RNA purification, which included ethanol precipitation of nucleic acids.
Following extraction, we eluted the samples with 12 μL RNase-free water and quantified the RNA concentration with the Qubit RNA HS Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) using a Qubit® Fluorometer.
3
Simultaneous DNA and RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were pelleted by centrifugation, and DNA and RNA were extracted using a protocol adapted from QIAGEN AllPrep DNA/RNA FFPE Kit (Cat# 80234), QIAamp DNA FFPE Tissue Kit (Cat# 56404) and miRNeasy FFPE kit (Cat# 217504). Briefly, cell pellets were resuspended in 240 μL Buffer PKD and 16 μL proteinase K (QIAGEN # 80234), lysed by vortexing, and centrifuged 20 min (15-30 min) at > 20,000 x g (room temperature). The supernatant was transferred to a new 1.5 mL centrifuge tube for RNA extraction, and the pellet was reserved for DNA extraction. The supernatant was incubated at 80°C for 15 min (on a thermal mixer at 300 rpm), and then centrifuged at 14,000 rpm for 2 min. The supernatant was transferred to a new 2.0 mL centrifuge tube and RNA was extracted using the miRNeasy FFPE kit (QIAGEN cat# 217504) according to the manufacturer’s instructions. The pellet containing the DNA was extracted using the QIAamp DNA FFPE Tissue Kit (QIAGEN cat# 56404) according to the manufacturer’s instructions. Both RNA and DNA kits utilize spin columns to wash and then elute nucleic acids.
4
Extraction of Total RNA from Cells and FFPE Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Total tissue RNA was extracted from FFPE tissue sections using the miRNeasy FFPE kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. Paraffin was removed from freshly cut FFPE tissue sections each up to 10-µm thick using deparaffinization solution using the miRNeasy FFPE kit (Qiagen, Inc.), and samples under went protease digestion at room temperature to release RNA from the sections, then short incubation (at 56°C for 15 min, then at 80°C for 15 min) to reverse formalin cross-linking of the released nucleic acids and DNase digestion to remove DNA. Total RNA (including miRNAs) was dissolved in 20 µl RNase-free water. RNA concentrations were measured using a NanoDrop-1000 (Thermo Fisher Scientific, Inc.) and RNA integrity was determined by 1.5% agarose gel electrophoresis.
5
Metastatic Melanoma miR-378a-5p Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA from formalin-fixed paraffin-embedded metastatic (27 cases) and in situ (13 cases) melanoma specimens collected at Istituti Fisioterapici Ospitalieri51 (link) were extracted using miRNeasy FFPE kit (Quiagen, Hilden, Germany) and retrospectively used for qRT-PCR analysis of miR-378a-5p expression. The ethics committee of the Regina Elena National Cancer Institute (Prot. CE/913/10) approved the use of human tissue samples according to biobanca criteria. Informed consent was obtained from all subjects.
6
miRNA Expression in Thyroid Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hematoxylin and eosin (H&E) stained slides from FFPE samples were reviewed by the pathologist, who confirmed the diagnosis and selected areas representative of both HCTC and normal tissue. Two to three cores (1 mm in diameter) of histologically confirmed HCTC and of normal tissue were obtained from each specimen. For all the patients, miRNA was extracted from FFPE samples of primary tumor and of normal thyroid tissue using Qiagen miRNeasy FFPE Kit (Quiagen, Hilden, Germany) according to the manufacturer's instructions. Quality of RNA and its size were assessed using the Agilent Small RNA kit (Agilent Technologies, California, USA). The expression of six miRNAs (miR-138, miR-183, miR-221, miR-222, miR-768-3p, and miR-885-5p) and U6 snRNA as endogenous control was determined using TaqMan MicroRNA Reverse Transcription Kit and specific TaqMan miRNA assays (TaqMan, Applied Biosystems, California, USA). Reactions for all miRNA assays and for all normal and tumor samples were performed in triplicate using the same amount of isolated RNA. Specific miRNAs, which were analyzed in our study, were selected based on data from the literature [17 (link), 26 (link)–28 (link)].
7
FFPE Tissue RNA Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four to five thick (10 μm) sections of FFPE tissue were used to extract total RNA. For the analysis of GD and GC samples, a pathologist assessed the slides to ensure the appropriate selection of tumor tissue and blocks with more than 50% tumor content per block. Deparaffinization solution (Qiagen, Hilden, Germany) was used to remove paraffin from FFPE tissue. For RNA extraction, we used the Qiagen miRNeasy FFPE kit (Qiagen) according to the manufacturer’s protocol. Total RNA concentration was determined by measuring the ratio of the absorbance at 260 and 280 nm using a NanoDrop™ 2000c Spectrophotometer (Thermo Fisher Scientific, USA). Total extracted RNA was stored at -70 °C until use.
8
RNA-Seq analysis of FFPE tissue samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NGS analysis was performed as follows: RNA was isolated from FFPE tissue sections using the Qiagen miRNeasy FFPE kit (Qiagen, Hilden, Germany) and purified using RNeasy MinElute cleaning (Qiagen). The total RNA concentration was measured using the Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), and the quality was checked using the 2100 Bioanalyzer with the RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). Library preparation was performed using QIAseq stranded total RNA library kit (Qiagen) with QIAseq Fast Select RNA Removal kit (Qiagen) for RNA depletion according to the manufacturer’s instructions and quality assessed by 2100 Bioanalyzer (Agilent). The libraries were run on the Illumina NextSeq 500 (Illumina) platform using NextSeq 500/550 High Output Kit v2.5 (75 Cycles) according to the manufacturer’s instructions. The analysis of the RNA sequencing data was processed using the Archer Analysis bioinformatics platform (v 6.2).
9
qRT-PCR Analysis of miRNA and mRNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cell lines and tissues using TRIzol (Invitrogen) or the miRNeasy FFPE kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. qRT-PCR was performed using the SYBR® Green PCR kit (Toyobo, Osaka, Japan) and the ABI PRISM® 7500 Sequence Detection System (Applied Biosystems, FosterCity, CA, USA). The primer sequences were as follows (Sangon, Shanghai, China): hsa-miR-301a-3p: forward, 5ʹ-ACACTCCAGCTGGGCAGTGCAATAGTATTGTC-3ʹ and reverse, 5ʹ-CTCAACTGGTGTCGTGGA-3ʹ; Smad4: forward, 5ʹ-TGCTGTTGCAAAGGCTGCTT-3ʹ and reverse, 5ʹ-GCTTCTGGCATAGCTGCATT-3ʹ. U6 or 18S rRNA was used as reference for miRNAs or mRNAs respectively. The primer sequences were as follows: U6: forward, 5ʹ-CTCGCTTCGGCAGCACA-3ʹ and reverse, 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ; 18S rRNA: forward, 5ʹ-CCTGGATACCGCAGCTAGGA-3ʹ and reverse, 5ʹ-GCGGCGCAATACGAATGCCCC-3ʹ. The qRT-PCR results, recorded as threshold cycle numbers (Ct), were normalized against an internal control. The 2-△△Ct method was used to quantify the relative levels of gene expression 23 (link). Each sample was analyzed in triplicate. The final measurement result was the average of the values obtained from 3 experiments.
10
Analysis of EZH2 and E-cadherin Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from the cultured cells by using miniBEST universal RNA extraction kit (Takara Bio, Inc.) and tissue samples using a miRNeasy FFPE Kit (QIAGEN) according to the manufacturer's instructions. Reverse transcription and RT-qPCR kits (Takara Bio, Inc.) were used to evaluate expression of EZH2 and E-cadherin. GAPDH expression was used to normalize for variance. The PCR Primers pairs used for each genes were:
EZH2 forward, 5′-CCGCAAGGGTAACAAAAT-3′;
EZH2 reverse, 5′-GGTAGCAGATGTCAAGGGA-3′;
E-cadherin, forward, 5′-TGTCCGCCCCGACTTGTCTCTC-3′;
E-cadherin reverse, 5′-GTCCTCTGGCCCCAGCCTCTCT-3′;
GAPDH, forward, 5′-CCCCGCTACTCCTCCTCCTAAG-3′;
GAPDH reverse, 5′-TCCACGACCAGTTGTCCATTCC-3′;
MALAT-1 forward, 5′-GAATTGCGTCATTTAAAGCCTAGTT-3′;
MALAT-1 reverse, 5′-GTTTCATCCTACCACTCCCAATTAAT-3′.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!