Is it possible to do RNA-seq analysis with bad 260/230 ratios?

Hi Alex

it depends, since this question depends on the RIN of the RNA. if the RIN is more than 8, no problemo. between 2 and 8, yes you can also do NGS but it's better to run 3'RNAseq only (and only have counts for the transcriptome), under RIN et 2, maybe you could forget, or just re-extract the samples...

all the best

fred

Mansoor Hayat

While having a poor 260/230 ratio in RNA samples can be suboptimal for RNA-seq analysis, it is still possible to perform the analysis with caution. The 260/230 ratio is a measure of RNA sample purity and indicates the presence of contaminants, such as salts, organic compounds, or other substances that can affect downstream applications. A "bad" 260/230 ratio typically implies contamination that may affect the quality of your RNA-seq data, but it doesn't necessarily make the analysis impossible.

Here are some considerations when working with RNA samples with poor 260/230 ratios:

Assess the Degree of Contamination: First, determine the extent of the contamination. If the ratio is slightly below the recommended range, it may still be manageable. However, if it's significantly below the expected value, it could pose more challenges.

Quality Control Steps: Conduct additional quality control steps, such as analyzing the RNA on an Agilent Bioanalyzer or TapeStation, which can provide more detailed information about RNA quality and integrity. This can help you make a more informed decision about whether to proceed.

Normalization and Data Analysis: When analyzing RNA-seq data from samples with poor 260/230 ratios, it's essential to use appropriate data normalization techniques to account for potential biases. Tools like DESeq2 or edgeR can help correct for any systematic variations in your data.

Reference Genomes and Annotations: Make sure you are using an appropriate reference genome and annotations for your species of interest. This can help mitigate potential issues caused by contaminants or low-quality RNA.

Replicates and Controls: If possible, include biological replicates and appropriate controls in your RNA-seq experiment. This can help account for variability and improve the robustness of your analysis.

Technical Replicates: Consider running technical replicates of your RNA-seq libraries to identify and account for any variability introduced during library preparation and sequencing.

Filtering and Preprocessing: Carefully preprocess your data, filtering out low-quality reads and potentially problematic samples. Tools like FastQC can help you assess the quality of your sequencing data.

Validation: Validate your findings through alternative methods, such as qRT-PCR, to ensure that the results obtained from RNA-seq are reliable despite the initial sample quality concerns.

Keep in mind that it's always preferable to start with high-quality RNA samples for RNA-seq analysis. However, if you have no other options and you are aware of the limitations, you can still attempt the analysis with caution and proper data handling. The specific approach you should take will depend on the extent of the 260/230 ratio issues and the goals of your experiment.

How should I go about evaluating cytoplasmic and nuclear Arginase-1 activity?

Does anyone know why I would have unexplained emission overlap for flow cytometry?

Does anyone know of a library for R that includes phi?

In SEM, how to interpret standardised regression weights and their significance?

Does anyone know how to add custom text to a violin plot in r, ggpubr made using the ggviolin line?

How do I interpret these correlations and p-values?

Is it possible to fuse Deep Learning Net and Clustering Algotrithm?

Are there models trained+evaluated on multiple datasets?

Are there models trained on both x-ray and ct scans for COVID-19 prediction?

When examining a series with different numbers of episodes per series, should I treat it as single sample, use harmonic mean or some other approach?

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

Any specific pipeline or parameter to analyse total transcriptomics study of brain tissue samples?

Why is the efficiency of viral mRNA electroporation in PBMC low?

Runs of nucleotides in Sanger sequencing?

Has anyone used the Illumina ITS1 primer sets?

Errors in DNA sequences?

What is the best concentration of cDNA to be used for qPCR?