Incubation of washed platelets with bacteria (5 × 108–109 platelets in 1:1 platelet to bacteria ratio, for three hours) was followed by centrifugation of the samples at 500 g for 10 minutes. The pellets were used for RNA isolation with a standard phenol-chloroform procedure. Briefly, centrifuged cells were resuspended in 500 µl Trizol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA), and added to Phase lock tubes (QuantaBio, Beverly, MA, USA) together with chloroform. All centrifugation steps were performed as previously described26 (link). The RNA pellet was air-dried and resuspended in 40 µl RNAse free water.
Phaselock tubes
Manufactured by Quantabio
Sourced in United States
PhaseLock tubes are a specialized laboratory equipment designed to facilitate the separation and extraction of aqueous and organic phases during liquid-liquid extraction processes. These tubes incorporate a physical barrier that helps to clearly delineate the interface between the two immiscible liquid phases, enabling efficient and precise phase separation.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Oligo dt beads, by Illumina (1 mentions)
D1000 kit, by Agilent Technologies (1 mentions)
Stranded mrna prep ligation kit, by Illumina (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Nanodrop 1000, by Agilent Technologies (1 mentions)
Rna 6000 nano, by Agilent Technologies (1 mentions)
Isopropanol, by Qiagen (1 mentions)
48 channel fragment analyser, by Agilent Technologies (1 mentions)
Phenol chloroform isoamyl alcohol, by Merck Group (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
4 protocols using phaselock tubes
1
Bacterial Interaction with Platelets
2
Conjunctival Transcriptome RNA-Seq Protocol
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Conjunctival tissue samples were collected into tubes containing RNAlater and were later homogenized using QIAzol lysis reagent (Qiagen) and ceramic beads (Precellys). Phase-lock tubes (Quantabio) were used to assist separation following chloroform-based phase separation of RNA before total RNA was extracted with the RNeasy Lipid Tissue Mini Kit (Qiagen). RNA quantification and quality assessment were performed with Nanodrop 1,000 and RNA 6,000 Nano (Agilent Bioanalyzer). Total RNA was diluted to 1 μg in 25 μL of RNase-free water prior to a polyA selection using Oligo(dT) beads from Illumina’s Stranded mRNA Prep Ligation kit. Enriched mRNA species were converted to full length complementary deoxyribonucleic acid, dual indexed, and then amplified using 10 PCR cycles. The final libraries were assessed using the D1000 kit on the TapeStation (Agilent) for quality as well as the High Sensitivity DNA Qubit for quantification. To prepare for sequencing, libraries were pooled together at equal molar before denaturing with sodium hydroxide and diluted to 1.5 pM in accordance with Illumina’s denaturation protocol for the NovaSeq6000. Sequence runs were performed on a NovaSeq6000 V1.5 SP flow cell and a 2′ 90 bp paired-end run.
3
RNA extraction from cell lysates
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Lysate aliquots (2–3 × 100 µL per sample) from the same sample were pooled, and if required RLT or QIAzol was added, to give a final lysis volume of 300 µL. Samples were transferred to 2 ml PhaseLock tubes (QuantaBio). For RLT lysates specifically, a half volume of phenol:chloroform:isoamyl alcohol (25:24:1 v:v:v; Sigma) was added before brief shaking and centrifuging at 4 °C. For both RLT and QIAzol samples, a half volume of chloroform:isoamyl alcohol (24:1 v:v) was added before shaking, a 3 min RT incubation and centrifugation at 4 °C. The aqueous phase was then transferred to a 1.5 ml Eppendorf tube and mixed with a 1.5 volume of isopropanol (Sigma). After thorough pipette mixing, the isopropanol mixture was applied to an RNeasy MinElute spin column and total RNA was extracted using the miRNeasy Micro Kit (Qiagen) with a DNase treatment. Samples were eluted in 14 µl nuclease-free water. RNA samples were assessed both quantitatively and qualitatively using the High Sensitivity Total RNA 15nt Analysis DNF-472 Kit on a 48-channel Fragment Analyser (Agilent). Total RNA yield was 0.31 ± 0.11 ng (mean ± SD) in the RLT lysate samples and 1.45 ± 0.51 ng in the QIAzol lysate samples; RNA integrity could often not be computed due to low input.
4
RNA Extraction from Cell Cultures
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After the challenge assay, supernatant was removed, and 2 mL Trizol Reagent (Invitrogen, Carlsbad, CA, USA) was added to the 25 cm2 tissue culture flask. After 5 min incubation with gentle agitation at room temperature, cell lysates were transferred to two PhaseLock tubes (Quantabio, Beverly, MA, USA), and 0.2 volumes of chloroform (v/v) was added to each tube. Tubes were shacked vigorously and centrifuged (12,000× g for 10 min at 4 °C). Aqueous phase containing RNA was transferred to a new tube, and RNA was precipitated by adding 0.5 volumes (v/v) of isopropyl alcohol. After centrifugation (12,000× g for 10 min at 4 °C), RNA pellets were washed with 70% ethanol and dried by placing them into a flow hood. Pellets were resuspended in 20 µL RNase-free water, and a step of RNA cleanup was performed, following the manufacturer’s instructions (RNeasy mini kit, Qiagen, Hilden, Germany). RNA was quantified by using Nanodrop, and RNA integrity (RIN) was determined with Bioanalyzer (RNA 6000 Nano Kit, Agilent Technologies, Santa Clara, CA, USA). RNA was then stored at −20 °C, until needed. Only RNA samples with a RIN > 8 were used in reverse transcription (RT) of mRNA into cDNA for subsequent gene expression microarrays [38 (link)].
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