To determine muscle protein, DNA and RNA concentrations (i.e. translational efficiency/capacity), ~15 mg of the medial portion of the right gastrocnemius muscle tissue of HRT-RES and LRT-RES animals was pulverized in liquid nitrogen and homogenized in 1 ml 0.2 M PCA. After centrifugation at 4°C at 11,600 g for 8 min to form a pellet and washing with 1 ml 0.2 M PCA (washing repeated twice), the pellet was resuspended in 800 μl 0.3 M NaOH and incubated at 37°C for 2 × 20 min to dissolve the pellet. The samples were gently vortexed before, in between and after the incubations. Total protein concentration was analysed as described above (see western immunoblot analyses). Thereafter, proteins were precipitated with 400 μl 1 M PCA before centrifugation at 4°C at 2400 g for 5 min. Next, 300 μl of 0.2 M PCA was added to the supernatant of each sample and centrifuged at 4°C at 2400 g for 5 min before removal of the supernatant for quantification of RNA by NanoDrop Lite Spectrophotometer (Thermo Scientific). The remaining pellet was resuspended in 1 ml of 2 M PCA and incubated at 70°C for 1 h before centrifugation at 4°C at 2400 g for 5 min. Next, 300 μl of 2 M PCA was added to the supernatant of each sample and centrifuged at 4°C at 2400 g for 5 min before removal of the supernatant for quantification of DNA by NanoDrop Lite Spectrophotometer (Thermo Scientific).
Nanodrop lite spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Australia, China, Switzerland, Canada, Japan, France
The NanoDrop Lite Spectrophotometer is a compact, portable device designed for the quick and accurate measurement of DNA, RNA, and protein concentrations. It uses a small sample volume to determine the concentration and purity of nucleic acid or protein samples.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (52 mentions)
Rneasy mini kit, by Qiagen (24 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (22 mentions)
Trizol, by Thermo Fisher Scientific (19 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (13 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (11 mentions)
Dnase 1, by Thermo Fisher Scientific (11 mentions)
Qiaamp dna mini kit, by Qiagen (10 mentions)
Primescript rt reagent kit, by Takara Bio (10 mentions)
Mirneasy mini kit, by Qiagen (10 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (9 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (9 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (8 mentions)
Quantitect reverse transcription kit, by Qiagen (8 mentions)
Tri reagent, by Merck Group (8 mentions)
Rnalater, by Thermo Fisher Scientific (8 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (8 mentions)
Iscript cdna synthesis kit, by Bio-Rad (7 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (7 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (7 mentions)
454 protocols using nanodrop lite spectrophotometer
1
Muscle Protein, DNA, and RNA Quantification
2
Gene Expression Analysis via RT-qPCR
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For gene expression analysis the total RNA extraction was performed by using Trizol® (ThermoFisher Scientific, Beverly, MA, USA) following the manufacturer protocol. After extraction, total RNA concentration was determined by using a NanoDropTM Lite spectrophotometer (ThermoFisher Scientific, Beverly, MA, USA). The integrity of total RNA was checked by using 1% agarose gel. After quality check, total RNA was reverse-transcribed into cDNA by using a Go Script Reverse Transcription kit (Promega, Madison, WI, USA) according to the manufacturer protocol.
Primers used for RT-qPCR of the target and housekeeping genes are described inTable 3 . RT-qPCR reactions were performed in QuantStudio® 3 (Applied Biosystems, Thermo Fisher Scientific).
The threshold cycle (Ct) values obtained were normalized (ΔCt) based on the Ct value of glyceraldehyde-3-phosphate dehydrogenase. Gene of interest relative expression was calculated by 2−ΔΔCt [24 ].
Primers used for RT-qPCR of the target and housekeeping genes are described in
The threshold cycle (Ct) values obtained were normalized (ΔCt) based on the Ct value of glyceraldehyde-3-phosphate dehydrogenase. Gene of interest relative expression was calculated by 2−ΔΔCt [24 ].
3
RNA Extraction from Rat Muscle Tissue
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Samples were randomly selected from rats in Study 1 and Study 2 experimental groups, and a total of 12 samples were used per extraction. From each sample, muscle tissue was crushed in a mortar with a pestle using liquid nitrogen and 200 mg was weighed on a scale. The weighed tissue was then transferred into a 1.5 mL tube and dissolved in a 1 mL Trizol (TRIzolTM Reagent, Thermo Fisher Scientific Inc., Waltham, MA, USA). This was followed by homogenization and incubation on ice for 5 min, and then centrifugation at 12,000× g at 4 °C for 15 min. Trizol was then discarded from the sample homogenate. Chloroform (200 µL) was added into the sample homogenate using a pipette, then incubated on ice for 15 min. Thereafter, the sample homogenate was centrifuged at 12,000× g for 15 min in order to achieve phase separation. The aqueous phase was then transferred into a 2 mL tube. Isopropanol (0.5 mL) was used to precipitate the RNA, followed by incubation on ice for 10 min. The sample was then centrifuged for 10 min at 12,000× g at 4 °C. After centrifugation, the supernatant was removed from the sample and the RNA sample pellet was air dried. RNase free water (20 µL) was then used to dissolve the extracted RNA pellet. This was followed by determination of RNA concentration and ratio using a nanodrop machine (NanoDropTM Lite Spectrophotometer, Thermo Fisher Scientific Inc.).
4
FFPE Tissue RNA Extraction Protocol
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RNA was extracted from FFPE samples using the AllPrep DNA/RNA FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A 3 µm section stained with hematoxylin and eosin (H&E) was histopathologically characterized at the Pathology Department at IPO-Porto and the number of 10 µm sections used for nucleic acid extraction varied from two to six, depending on the size of the limited area (up to 6 cm2) enriched in “normal”, distant from the tumor whenever possible, or tumoral cells. Using a sterile single-use scalpel, the area was macrodissected into a 1.5 mL microcentrifuge tube containing 1 mL of deparaffination reagent D-limonene (Santa Cruz, Dallas, TX, USA) by scratching. The kit instructions were followed throughout the remaining procedure.
The resulting RNA was quantified using the NanoDropTM Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), its quality was assessed by measuring the optical density (OD) 260/280 ratio, and it was kept at −20 °C until further processing.
The resulting RNA was quantified using the NanoDropTM Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), its quality was assessed by measuring the optical density (OD) 260/280 ratio, and it was kept at −20 °C until further processing.
5
Detecting Morphological Markers by qRT-PCR
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Molecular markers associated with morphological alterations were detected by qRT-PCR. Total RNA was extracted from the leukocyte samples with the miRNeasy mini kit reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA quality and amount were determined using a Nanodrop TM Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and by gel electrophoresis. For the analysis, 500 ng of total RNA was retrotranscribed using the iScriptTM cDNA Synthesis Kit (BioRad, Hercules, CA, USA) following the manufacturer’s instructions. For gene expression analysis, 5 ng of cDNA was processed in duplicate in a Rotor-Gene Q real-time machine (Qiagen, Hilden, Germany) using the SsoAdvanced Universal SYBR® Green SuperMix (BioRad, Hercules, CA, USA). The PCR conditions were as follows: 30 s of initial denaturation (95–98°) and then 40 cycles at 95 °C for 5 s, 60 °C for 20 s. To assess product specificity, a melting curve analysis from 65 °C to 95 °C was performed. The gene transcript values were normalized using GAPDH as a reference gene. The relative quantification of the samples was performed by the Rotor-Gene AssayManager® v1.0 (Qiagen, Hilden, Germany). The complete list of the primer sequences is shown in Table 2 .
6
FFPE Nucleic Acid Extraction Protocol
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The nucleic acids were extracted from up to 6 cm2 of macrodissected FFPE areas, enriched in “normal” or tumor cells, using the AllPrep® DNA/RNA FFPE Kit (Qiagen, Hilden, Germany) following the manufacturer instructions and quantified by NanoDropTM Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The quality was assessed by measuring the optical density (OD) 260/280 ratio and samples were kept at −20 °C until further processing.
7
Quantitative Real-Time PCR Analysis of Gene Expression
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Total cellular RNA was extracted with a Maxwell RNA purification kit (Promega, Madison, WI, USA), and quantified using a NanoDropTM Lite spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). RNA purity was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Five micrograms of total RNA from each sample were reverse transcribed using the SuperScript® VILOTM cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA). Primers designed according the Roche Universal Probe Library (UPL) were validated with Stratagene cDNA mix (Agilent Technologies) (Supplementary Table 1 ). RT-qPCR reactions were performed using a LightCycler® 480 Probes Master (Roche Life Science, Basel, Switzerland). Samples were subjected to an initial denaturation step (5 min, 95 °C), followed by 45 PCR cycles (10 s, 95 °C, then 30 s, 60 °C) and a final cooling step (40 °C, 30 s). All reactions were run concomitantly in triplicates, and results analyzed using the Cycle threshold (Ct) values. The geometric Ct mean of human ACTB, YWHAZ, RPL13A, EF1A, and GAPDH genes were used as endogenous control to normalize the expression of target genes: ΔCt = “Ct target” − “Ct reference”.
8
Genomic DNA Extraction from CRE/ESBL Isolates
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Genomic DNA was extracted from all CRE/ESBL-producing isolates using the Zymo Research Genomic DNATM–Tissue MiniPrep Kit (Zymo Research Corp, Irvine, CA, USA) according to the manufacturer’s instructions. The quality and quantity of DNA was determined using NanodropTM-Lite spectrophotometer (Thermo Scientific, Walton, MA, USA). Pure DNA samples were stored at −20 °C for future use.
9
Extraction and Quantification of Plant RNA
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Plant tissues were ground in liquid nitrogen using a mortar and pestle. For isolating the RNA from shoots and roots, 0.1 g of tissue was used, and the RNA was isolated using an RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). For the seeds, 0.2 g of ground tissue was used for RNA isolation according to the protocol described by Wang et al. [59 (link)], with some modifications. The changes to the protocol included a larger volume of RNA extraction buffer (600 µL), a larger volume of 20% sodium dodecyl sulfate (SDS, 30 µL), and longer RNA precipitation in ethanol (overnight at −20 °C). The quality and quantity of isolated RNA were checked via agarose gel electrophoresis and spectrophotometric measurement using a NanoDropTM Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
10
Cloning, Expression, and Purification of Targeted Toxin
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The targeted toxin PE24mut was cloned, expressed and purified as described previously 9 . The targeted toxin EGF-PE24mutΔREDLK, lacking the C-terminal REDLK sequence, was generated as follows. The gene of the toxin domain PE24mutΔREDLK including a stop codon was codon optimized for expression in E.coli, synthesized (Geneart, Regensburg, Germany) and cloned via the XbaI restriction site into the expression vector pHOG21 C-terminally to the sequence of the human EGF ligand (Fig. 1 A). The pHOG21 vector contains a pelB leader sequence for periplasmatic expression and a human c-myc tag and a hexahistidine tag for detection and purification, respectively. EGF-PE24mutΔREDLK was periplasmatically expressed in E.coli bacteria and purified by immobilized affinity chromatography (IMAC) using HiTrapTM Chelating High Performance columns (Sigma-Aldrich, St. Louis, MO, USA) as described 9 . The targeted toxin was stepwise eluted from the columns by increasing imidazole concentrations (40 mM - 250 mM) and dialyzed against PBS. Protein concentration of the purified targeted toxin was determined by NanoDropTM Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
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