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To calculate the relative gene expression using the ΔΔCt (delta-delta Ct) method in qPCR, follow these steps: Key Components: 1. Ct values: The cycle threshold values from the qPCR data. 2. Housekeeping gene: Used to normalize the expression of target genes. 3. ΔCt (delta Ct): Normalization against the housekeeping gene. ΔCt=Ct target − Ct housekeeping 4. ΔΔCt (delta-delta Ct): Comparison to the control sample. ΔΔCt=ΔCt treatmen t− Δct control 5. Fold change in expression: Fold change=2−ΔΔCt Example Process: 1. Average the triplicates: Compute the mean Ct values for each gene (target and housekeeping) from the triplicate qPCR runs. 2. Calculate ΔCt: For each sample: ΔCt=Mean Cttarget−Mean Cthousekeeping 3. Choose your control sample: Use the control group to calculate the baseline ΔCt: ΔCt control= Mean Ct target−Mean Ct housekeeping 4. Calculate ΔΔCt: For each treatment: ΔΔCt=ΔCttreatment−Δctcontrol 5. Determine fold change: For each treatment, use: Fold change=2−ΔΔCt Please let me know if this was useful.

I'm glad you found it helpful!

Thank You

As far as I know, it is not possible in Gen5, but you can do it using third-party software. But it is recommended to keep all required control in each ELISA plate

Thank you

Hi DeepNithun For qPCR-HRM analysis, it’s common to see variations in the HRM curve if there are differences in sample quality, concentration, or minor technical differences in handling. Using SYTO 9 dye should generally work, but if other studies in your field are using EvaGreen with a Bio-Rad instrument, this could be due to slight compatibility and sensitivity differences with dyes and instruments. EvaGreen is often preferred in HRM due to its higher affinity for double-stranded DNA, which can sometimes lead to more consistent results in distinguishing variant curves. Here are a few tips: 1. Recheck DNA concentration and purity: Small differences can impact the melting curve. 2.Ensure consistent sample handling: Variations in pipetting or mixing can cause curve shifts. 3.Consider switching to EvaGreen: Since it’s commonly used in your research area, it might offer improved consistency with your samples. If you still see distinct variant curves, it might indicate true sample heterogeneity. Feel free to provide more details if you continue to have issues. Best of luck with your research!

@@LearnInnovativelywithMe Understood sir. Thank you for your swift response sir.

I will upload shortly, but it is not possible at this time.

Dear JoePenot, It seems that the XLe800 device is not showing up under "Device Manager > COM and LPT," although it appears in "Settings > Devices." Could you please: Check the connection cord or wire to ensure that it is properly plugged in. Confirm that the instrument is powered on and functioning correctly. These steps can help resolve connectivity issues. If the issue persists, I recommend contacting the service engineer for further assistance.

Dear nestorsoler, Thank you for your inquiry about the Gen5 software. You can obtain the Gen5 software by following these steps: Visit the Manufacturer's Website: Go to the official website of the manufacturer, BioTek Instruments, where you can find the latest version of Gen5 software. web link: For Gen5: www.agilent.com/cs/library/software/public/Gen5-v3.16.10-software-5320200-REV-AP-agilent.zip For Gen6: www.agilent.com/cs/library/software/public/1950200-REV-D.zip Contact Sales or Support: If you require a specific version or have questions about licensing, I recommend reaching out to their sales or customer support team for guidance. Check for Institutional Access: If you are affiliated with an institution or organization, they may have a license for the software. You can check with your IT department or lab manager for more information. If you need further assistance, feel free to ask!

@@avishekshaw3709 In Gen5 software, if you begin with calibrating just one well, you can still proceed smoothly with the rest of the plate. Here’s how: 1. **Initial Calibration**: After placing your calibration standard in one well and performing the calibration, make sure to save the calibration data. Gen5 software will store the calibration results, and you can apply them to the entire plate. 2. **Assigning Calibration to All Wells**: In the plate layout section of Gen5, there’s an option to apply the calibration results across all wells. Go to the 'Plate Layout' or 'Protocol' section, where you can replicate the calibration data to all the other wells that will contain your samples. 3. **Running the Full Plate**: Once the calibration has been applied, you can insert the plate with all your samples. The software will use the saved calibration data for all wells, ensuring uniform analysis. 4. **Verify Calibration**: Before proceeding with measurements, it’s a good practice to double-check the calibration in the 'Data Reduction' or 'Analysis' tab to confirm that it has been correctly applied across the entire plate. By following these steps, you can perform calibration on a single well and still ensure accurate results for all the other wells in your plate. Gen5 is designed to simplify this process by allowing you to easily apply calibration settings across multiple wells.

Thank you for reaching out! The functionalities you're referring to are not default functions in PowerPoint but are instead created using VBA (Visual Basic for Applications) code. You can easily create a Macro-enabled PowerPoint (PPTM) in your system by using the provided VBA code. Make sure to enable macros and adjust related settings to allow the code to run properly. If you prefer not to create it yourself, no worries! You can use the Macro-enabled PowerPoint I’ve provided, which already contains the necessary VBA code. Let me know if you need any further assistance!

Glad it was helpful!

Hi! Thanks for your comment. I currently don't use Quad Studio 7 Pro, but importing a plate map usually follows a similar process in most software. You typically need to format your plate map in a compatible spreadsheet format (like Excel or CSV), ensuring the wells are correctly labeled. Then, look for an option within the software to import the file under the 'Plate Setup' or 'Layout' tab. If I come across any specific steps for Quad Studio 7 Pro, I’ll share them. Meanwhile, I suggest checking the user manual or support forums for more detailed guidance. Hope this helps!

steps: 1. Create Your Plate Map: Prepare your plate layout in a spreadsheet (Excel or CSV format), ensuring each well is labeled with the appropriate sample, control, or blank. 2. Find the Import Option: In Quad Studio 7 Pro, look for the 'Plate Setup' or 'Layout' section. There should be an option to import your plate map. It might be under 'File' or 'Import Plate Layout.' 3. Select Your File: Choose the file you created and follow any prompts to map the wells correctly. I recommend checking the software manual or help section for detailed steps tailored to Quad Studio 7 Pro. Hope this helps! Feel free to reach out if you have more questions.

Please follow these step I hope this may help you But please varify the results on your basis. Good Luck Step 1: Gather Ct Values First, you'll need to obtain the Ct (threshold cycle) values for both your reference gene (GAPDH) and your target genes under each condition: DMSO (control), compound A at 50 μg/mL, and compound A at 100 μg/mL. Step 2: Calculate Δct The ΔCt value is calculated by subtracting the Ct value of the reference gene (GAPDH) from the Ct value of the target gene for each sample. ΔCt=CtTarget Gene - CtReference Gene For each condition (DMSO, 50 μg/mL, 100 μg/mL), calculate Δct as follows: • DMSO (control): ΔCTDMSO = CtTarget Gene, DMSO - CtGAPDH, DMSO • Compound A at 50 μg/mL: ΔCt50 μg/mL = CtTarget Gene, 50 μg/mL - CtGAPDH, 50 μg/mL • Compound A at 100 μg/mL: ΔCt100 μg/mL = CtTarget Gene, 100 μg/mL - CtGAPDH, 100 μg/mL Step 3: Calculate ΔΔCt The ΔΔCt value is calculated by subtracting the ΔCt value of the control (DMSO) from the ΔCt value of each treated sample. ΔΔCt=Δct Treatment - ΔCtControl For each treatment, calculate ΔΔCt as follows: - • Compound A at 50 μg/mL: ΔΔCt50 μg/mL = ΔCt50 μg/mL - ΔCTDMSO • Compound A at 100 μg/mL: AAC100 μg/mL =ΔCt100 μg/mL -ΔCtDMSO Step 4: Calculate Relative Gene Expression Finally, the relative expression level of the target gene can be calculated using the formula: Relative Expression = 2-ΔΔCt This value represents the fold change in gene expression of the treated samples compared to the control (DMSO). Example: Suppose you have the following Ct values: DMSO (control): Ct Target = 22, Ct_GAPDH = 18 Compound A at 50 μg/mL: Ct_Target = 20, Ct_GAPDH = 18 Compound A at 100 μg/mL: Ct Target = 19, Ct_GAPDH = 18 Then, calculate as follows: ΔCt for DMSO: ΔCTDMSO = 22-18=4 Δct for Compound A (50 μg/mL): ΔCt50 μg/mL=20-18=2 •ΔCt for Compound A (100 μg/mL)): ΔCt100 μg/mL = 19-18=1 ΔΔCt for 50 μg/mL: ΔΔCt50 μg/mL = 2-4=-2 ΔΔCt for 100 μg/mL: ΔΔC100 μg/mL =1-4=-3 Relative Expression for 50 μg/mL: 2-ΔΔCt-2-(-2) = 2 2 = 4 fold increase Relative Expression for 100 μg/mL: 2-ΔΔCt-2-(-3)=2 3=8 fold increase

Thank you

Thank you so much for your kind words! I’m really glad to hear that you found the content informative and helpful. Your support and feedback mean a lot to me. I'll definitely keep working hard to bring more valuable content. If you have any more questions or suggestions, feel free to let me know!

Thank you for your kind words about the video! I’m glad you found it helpful. To answer your questions: Using One-Way ANOVA with Multiple Groups: Yes, you can use a one-way ANOVA to analyze data from more than two groups. In your case, with five groups, one-way ANOVA is appropriate for comparing the means across these groups to determine if there are any significant differences. Analysis with Two Replicates: The minimum requirement for robust statistical analysis is typically three replicates per group. With only two replicates, the results may not be as reliable, and the statistical power to detect differences may be limited. If you only have two replicates, you could consider: Non-parametric Tests: Use non-parametric tests like the Mann-Whitney U test if comparing two groups or the Kruskal-Wallis test for more than two groups. These tests are less reliant on assumptions about the data distribution and can be used with small sample sizes. Bootstrap Methods: Employ bootstrap techniques to estimate confidence intervals and assess the variability in your data. This method can help in understanding the potential variability in the absence of sufficient replicates. Increasing Replicates: If possible, adding more replicates to your study would provide more reliable data and enable the use of more robust statistical tests. I hope this helps with your analysis! Feel free to reach out if you have more questions

Thank you for the compliment on the video! I’m glad you found it valuable. If you’re interested in learning more, you can connect with me through the email learninginnovatively@gmail.com for any questions or to discuss specific topics. Feel free to reach out through any of these channels. I’m here to help and look forward to connecting with you!

@@LearnInnovativelywithMe sir thanks for the reply,. Please make a video on biotek multimode reader Usage through gen5, grateful for the efforts you are putting sir

Thanks a lot!

Yes, in PowerPoint, you can export figures as PNG files without sacrificing quality by following these steps: Save As PNG: Select the slide or the figure you want to export. Go to File > Save As. Choose the location where you want to save the file. In the Save as type dropdown menu, select PNG Portable Network Graphics Format. Click Save. Choose Just This One if you’re saving only the selected slide or Every Slide if you want to export all slides as PNG files. Set High Resolution: By default, PowerPoint exports images at a standard resolution, but you can increase this by modifying the registry (for advanced users). My TH-cam Tutorial Link For this th-cam.com/video/RuH_yA_mvrA/w-d-xo.htmlsi=2N5rX8zmgU0r6MG5 Windows Registry Method: Open the Windows Registry Editor (type regedit in the Start menu search and hit Enter). Navigate to HKEY_CURRENT_USER\Software\Microsoft\Office\<version>\PowerPoint\Options (replace <version> with your version of Office, e.g., 16.0 for Office 2016). Right-click in the right pane, select New > DWORD (32-bit) Value. Name it ExportBitmapResolution. Double-click the new value and set it to one of the following (in decimal): 96 for 96 DPI 150 for 150 DPI 300 for 300 DPI 600 for 600 DPI 1200 for 1200 DPI Click OK and close the Registry Editor. Restart PowerPoint to apply the changes. Use High-Resolution Export Tools: For even more control over image quality, consider using external tools or software like Adobe Illustrator or dedicated image editing programs. These tools allow for high-quality exports and further adjustments. By following these steps, you can ensure that your PNG files retain high quality when exported from PowerPoint.

Thanks for the visit

I haven’t tried comparing UMAP and Concordex myself, but it sounds like an interesting comparison. Each method has its strengths, so exploring how they perform on your data could provide valuable insights. If you’ve done any experiments or have thoughts on how they differ, I’d love to hear about your findings!

​@@LearnInnovativelywithMe My master's thesis is about comparing UMAP and Concordex. I've been looking for help, but if I graduate I'll send you my thesis!

Thank you for your interest! I’ll definitely cover SNP analysis in a future video, but I’ll be focusing on finishing the current series first. Stay tuned for updates, and I’ll be sure to include a detailed tutorial on SNP analysis once this series is complete. Thanks for your patience!

Hi sir, thank you for ur tutorials, It's very impressive. For a long time I have been waiting for SNP's analysis but no single video available on youtube. Kindly make SNP's analysis and interpretation with or without software tools. I'm eagerly waiting.

Yes, you're correct. In qPCR using TaqMan assays, it is essential to run the endogenous control (housekeeping gene) and the target of interest in separate wells. This separation ensures accurate measurement of the relative expression levels, as it prevents any potential interference between the two assays and allows for a more reliable comparison. Running them in the same well could lead to inaccurate results due to potential signal overlap or differences in amplification efficiency.

To download and install GraphPad Prism, follow these steps: Download GraphPad Prism Visit the GraphPad Website: www.graphpad.com/ Go to the GraphPad Prism website. Download the Trial or Purchase: If you want to try the software before purchasing, click on the "Download Free Trial" button. If you are ready to purchase, click on "Buy Now" and follow the instructions to complete the purchase and download. Choose Your Operating System: Select the appropriate version for your operating system (Windows or macOS). Install GraphPad Prism For Windows: Run the Installer: Locate the downloaded .exe file and double-click it to start the installation process. Follow the Installation Wizard: Click "Next" to proceed through the installation steps. Read and accept the license agreement. Choose the installation location or use the default path. Click "Install" to begin the installation. Complete the Installation: Once the installation is complete, click "Finish" to exit the wizard. Activate the Software: Open GraphPad Prism from the Start menu. Enter your license key if you purchased the software, or follow the instructions to use the trial version. For macOS: Open the Disk Image: Locate the downloaded .dmg file and double-click it to open the disk image. Drag to Applications: Drag the GraphPad Prism icon to the Applications folder. Open GraphPad Prism: Go to your Applications folder and double-click GraphPad Prism to open it. Activate the Software: Enter your license key if you purchased the software, or follow the instructions to use the trial version. If you encounter any issues during installation, you can refer to the GraphPad Prism support page for troubleshooting tips and additional assistance.

Thank you for the update. Since shortening the path and reinstalling FlowJo did not resolve the issue, and the problem persists with all plugins, there might be another underlying issue. Here are a few additional steps you could try: Verify Plugin Compatibility: Ensure that all plugins are compatible with your version of FlowJo. Sometimes, incompatibility issues can cause errors. Check for Software Updates: Make sure you have the latest updates for both FlowJo and the plugins. Sometimes, updating to the latest version can resolve compatibility issues. Review Logs: Look at the FlowJo logs or error messages for more detailed information about the problem. This can provide clues as to what might be causing the issue. Contact Support: If the issue persists, consider reaching out to FlowJo support for more detailed assistance. They might be able to provide specific guidance based on your setup and error messages. If you have any other details or screenshots that might help in diagnosing the problem, please share them. We're here to help!

www.bdbiosciences.com/content/dam/bdb/products/global/reagents/immunoassay-reagents/cba/cba-kits/551xxx/5518xx/551811_base/pdf/551811_Book_Website.pdf

www.bdbiosciences.com/content/dam/bdb/products/global/reagents/immunoassay-reagents/cba/cba-kits/552xxx/5523xx/552364_base/pdf/23-12720.pdf

@@LearnInnovativelywithMe thank you so much . But i need Technical plz , because im ingénieur fse

Adding a line of significance or a significance marker to a graph can typically be done in various data visualization tools and software. Here's how you can do this automatically in some popular tools: 1. GraphPad Prism Steps to Add a Significance Line in GraphPad Prism: Create Your Graph: Enter your data and create the desired graph (e.g., bar graph, scatter plot) using GraphPad Prism. Add a Line of Significance: Go to the "Format Graph" tab. Choose "Lines" from the menu. You can add a horizontal line by setting the "Y-value" at the significance threshold or use the "Add Annotation" feature to manually draw and position the line. To add a line representing a p-value or significance level, you can use the “Draw” feature in the "Format Graph" window. Customize the Line: Adjust the line style, color, and thickness as needed. Add a Legend or Annotation: Include a text annotation or legend explaining the significance level or what the line represents.

To get Gen5 software, follow these steps: Visit the Manufacturer's Website:www.agilent.com/en/support/biotek-software-releases Go to the BioTek Instruments website or the relevant site for your specific instrument if BioTek has a dedicated page for Gen5. Navigate to the Gen5 Software Page:www.agilent.com/en/support/biotek-software-releases Look for the “Software” or “Downloads” section on the website. You may find a direct link to Gen5 software under the software downloads or support section. Register or Log In: You may need to register for an account or log in to access the download links. BioTek often requires users to create an account to download software. Download the Software: Select the version of Gen5 you need and download it. Ensure you choose the version compatible with your operating system. Install the Software: Once downloaded, open the installer file and follow the installation instructions. You might need administrative privileges to complete the installation. Contact Support: If you have trouble finding or downloading the software, contact BioTek customer support for assistance. They can provide you with direct links or further guidance. If you have a specific model or need for Gen5, make sure to download the version that is compatible with your BioTek equipment.

Thank you for appreciating. I am working on other tutorial of the RNA seq analysis series. I will upload it soon.

Thanks a lot Sir! ❤❤🎉🎉

Thank you

In real-time PCR, "target" refers to the specific DNA sequence or gene that you're interested in amplifying and quantifying. This could be a gene associated with a disease, a genetic marker, or any specific DNA sequence you want to study. On the other hand, "sample" refers to the biological material or substance from which you're extracting DNA to analyze. This could be anything from blood, tissue, saliva, or even environmental samples like soil or water. So, in real-time PCR, you're essentially trying to detect and quantify the amount of a specific DNA target present in your sample. This technique is incredibly valuable in various fields such as medical diagnostics, environmental monitoring, and genetic research. Hope this helps clarify things for you! If you have any more questions, feel free to ask.