Nanodrop nd 2000 spectrophotometer

To minimize wounding- and dehydration-induced gene expression, leaf samples were quickly harvested and immediately frozen in liquid nitrogen. Each combination of time and treatment (with and without infestation) was represented by three biological replicates. Frozen samples were ground to a fine powder in liquid nitrogen with a pestle and mortar. Total RNA was extracted from 100 mg of each leaf sample using a plant RNA isolation kit (Axygen, Hangzhou, China) according to the manufacturer’s instructions. RNA concentration and purity were determined using a NanoDrop TM ND-2000 spectrophotometer (Thermo Scientific, Wilmington, DE, United States), and the integrity of RNA was also assessed by 1% agarose gel electrophoresis and ethidium bromide staining. First-strand cDNA was synthesized from 200 ng of RNA using a First-Strand cDNA Synthesis Kit (Bio-Rad, Hangzhou, China) according to the manufacturer’s instructions.

Total RNA was isolated from tumor tissues and purchased cell lines using TRIzol (Life Technologies) according to supplier’s instructions. RNA concentration was measured with a NanoDrop ND-2000 spectrophotometer (Life Technologies). Reverse transcription was performed with random primers using the First Strand cDNA synthesis kit (Takara, Otsu, Shiga, Japan) according to the manufacturer’s instructions. The quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the SYBR Select Master Mix (Applied Biosystems, cat: 4472908) on ABI 7500 system (Applied Biosystems, CA, USA) according to the manufacturer’s instructions. β-actin was measured as an internal control. The relative expression fold change of mRNAs was calculated by the 2^−ΔΔCt method. After the reverse transcription, 0.5 μl of the complementary DNA was used for subsequent qRT-PCR reaction. The primer sequences were as follows: β-actin: 5’-GAAATCGTGCGTGACATTAA-3’ (forward), 5’-AAGGAAGGCTGGAAGAGTG-3’ (reverse); GAS5: 5’-TGAAGTCCTAAAGAGCAAGCC-3’ (forward), 5’-ACCAGGAGCAGAACCATTAAG-3’ (reverse); SNORD78: 5’-GTGTAATGATGTTGATCAAATGTCTGAC-3’ (forward), 5’-CACATTACTACAACTAGTTTACAGACTGG-3’ (reverse).
For cell expression and tumor samples, each sample was run in triplicate. qRT-PCR results were analyzed and expressed relative to CT (threshold cycle) values, and then converted to fold changes.

The TRIzol reagent (Invitrogen, China) was applied to extracted total RNA. RNA concentration was examined with a NanoDrop ND-2000 spectrophotometer (Life Technologies). Quantitative real-time PCR (qPCR) was carried out by the application of the SYBR Green quantitative PCR kit (Takara, Hangzhou, Zhejiang, China) using the 7500 Real-Time PCR System. The results were normalized to the expression of GAPDH. The primers were as follows: GAPDH sense 5′- ACAACTTTGGTATCGTGGAAGG-3′, reverse 5′- GCCATCACGCCACAGTTTC-3′ and PLXDC2 sense, 5′- CCAGTTTCAGTTCGCCGATG-3′, reverse 5′- TGTCTACCGCCTTGAGAAAGT-3′. The qRT-PCR data were analyzed and calculated using the 2^−ΔΔCt methods.

Total RNA was extracted from various tissues and cells using Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instruction. The quality of total RNA was checked by 1% agarose gel electrophoresis. The concentration of total RNA was determined using a Nanodrop ND-2000 spectrophotometer (Thermo Electron Corporation, USA). The first-strand cDNA was generated using the Revert Aid™ M-MLV Reverse Transcriptase Kit (Promega, USA) following the manufacturer’s instructions. The cDNA products were stored at –20°C.

Extraction of the total RNA was done via the RNA easy Extraction Kit (Qiagen) according to the manufacturer’s guidelines. Both yield and purity were assessed at 260 and 280 nm respectively using a Nanodrop ND-2000 spectrophotometer (Thermo Electron). A total amount of 1 μg RNA was used for cDNA synthesis by Viva 2-steps RT-PCR Kit according to the manufacturer’s instructions.
Quantitative PCR using Thermo Scientific Verso 1-step RT-PCR Ready-Mix kit (Applied Biosystems, Foster City, CA, USA) was conducted to analyze the levels of the mRNA of the target genes which quantified relatively in relation to the expression rate of β-actin. The primers of amplification include IL-6 F: 5′-GCCT TCTTGGGACTGATG-3, R: 5′-TGGTCTGTTGTGGGTGGT-3′, TNF-α F: 5′-GCTGAGGTTGGACGGATAAA-3′, R: 5′-AAAATCCTGCCCTGTCACAC -3′, and TGF-β F: 5′-TGGCGTTACC TTGGTAACC- 3′, R: 5′- GGTGTTGAGCCCTTTCCAG- 3′, and B-actin, F: 5-d TCCCTGAAGTACCCCATGGAG-3′, R: 5′-d TTGGCCTTGGGGTTCAGGGGG-3.

Total RNA was extracted using TRIzol Reagent (TRIzol^® Plus RNA Purification Kit; Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. The integrity and purity of RNA were determined by electrophoresis on a 1% agarose gel and a Nanodrop ND-2000 spectrophotometer (Thermo Electron Corp., Waltham, MA, USA), respectively. Total RNA was reverse-transcribed to cDNA with a PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan) and stored at −20 °C before analysis.