Gene and miRNAs expression levels were validated by qRT-PCR on both training (array set) and validation set of patients. qRT-PCR have been performed using Sybr Green protocol (Qiagen, Milano, Italy) on an Applied Biosystems 7900HT instrument. Experiments were run in triplicate, using 384-well reaction plates in an automatic liquid handling station (epMotion 5075LH; Eppendorf, Milano, Italy). Raw data was generated with Sodium dodecylsulphate (SDS) Relative Quantification software (version 2.3; Ambion-ABI), data were normalized using the geometric mean of the four independent housekeeping controls (for miRNAs: RNU6B, SNORD61, SNORD72, SNORD68; for genes: ACTB, B2M, PPIA and HPRT1). Two-sided Student's t-test were used to verify among groups mean differences; P-value ≤0.05 was considered statistically significant.
Epmotion 5075lh
Manufactured by Eppendorf
Sourced in Italy, Germany
The EpMotion 5075LH is a liquid handling workstation designed for precise and automated pipetting. It features a high-precision pipetting system, a temperature-controlled workspace, and a variety of accessories for customizable liquid handling tasks. The EpMotion 5075LH is capable of performing a range of liquid handling operations, including aspirating, dispensing, and mixing, with a focus on accuracy and reproducibility.
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5 protocols using epmotion 5075lh
1
Validating Gene and miRNA Expression
2
miRNA Expression Quantification by qRT-PCR
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miRNA expression levels were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR), using Sybr Green protocols and commercially available assays (Qiagen) on an Applied Biosystems 7900HT instrument. Experiments were run in triplicate, using 384-well reaction plates in an automatic liquid handling station (epMotion 5075LH; Eppendorf). Analysis was conducted as previously described [21 (link)], using 4 independent housekeeping genes (SNORD 72, SNORD 95, RNU 6B, and RNU 5A). Cycles' threshold for selected genes was in line with those obtained in the past for snap frozen tumor tissue in our experimental condition, which is an indirect measure of the good quality of RNA used for the analysis. To verify mean differences among groups, normalized PCR data were analyzed using Wilcoxon rank test using GraphPad Prism version 6.03 for Windows (GraphPad Software, La Jolla, California, USA, http://www.graphpad.com/ ). A two-sided P value < 0.05 was considered statistically significant.
3
ELISA-based IBV Antibody Titration
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A commercial kit of ELISA IBV Ab Tests (IDEXX Laboratories) was used to determine the titer of anti-IBV–specific IgY in broiler serum. Particular stages of the tests were performed with an Eppendorf epMotion 5075 LH automatic pipetting station (Eppendorf, Germany), a BioTek ELx405 automatic multi-well plate washer (BioTek), and a BioTek EL×800 plate reader. The mean geometric titer of antibodies and CV% were computed for each group in each sampling period.
4
Validating Gene and miRNA Expression
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Gene and miRNAs expression levels were validated by qRT-PCR on both training (array set) and validation sets of patients. qRT-PCR have been performed using Sybr Green protocol (Qiagen, Milano, Italy) on an Applied Biosystems 7900HT instrument (Waltham, MA, USA). Experiments were run in triplicate, using 384-well reaction plates in an automatic liquid handling station (epMotion 5075LH; Eppendorf, Milano, Italy). Raw data was generated with Sodium dodecylsulphate (SDS) Relative Quantification software (version 2.3; Ambion-ABI), data were normalized using the geometric mean of the four independent housekeeping controls (for miRNAs: RNU6B, SNORD61, SNORD72, SNORD68; for genes: ACTB, B2M, PPIA and HPRT1). Two-sided Student’s t-test were used to verify between groups’ mean differences, then the p-value underwent Benjamini–Hochberg FDR-controlling procedure, and only comparison with adjustedp-value ≤ 0.1 was considered statistically significant and reported in Table 2 .
5
ELISA-based Serological Monitoring of Poultry Pathogens
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A commercial kit of ELISA Ab Tests (IDEXX Laboratories, USA) was used to determine the titer of anti-IBV, anti-REO, anti-FAdV, and anti-IBDV speci c IgY in broiler serum. Particular stages of the tests were performed with an Eppendorf epMotion 5075 LH automatic pipetting station (Eppendorf, Germany), a BioTek ELx405 automatic multi-well plate washer (BioTek, USA), and a BioTek ELx800 plate reader. Sample to positive (S/P) ratio, plus/minus standard deviation (SD) were computed for each group in each sampling period.
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