With a myriad of liquid handling devices available in the market ranging from a simple pipette to a full-scale automated workstation, making the right choice can be daunting. According to Kay Chang, the Product Manager of Liquid Handling at Blue-Ray Biotech, one should consider certain key specifications while buying a liquid-handling solution:

• Size
• Throughput
• Channel Number
• Labware Compatibility
• Accuracy and Precision
• Volume Range

However, the importance of other factors, such as cost, ergonomics and extent of automation should not be overlooked. Begin by asking the following basic questions:

1. Does your lab have enough room to install the proposed system? You should check with the manufacturer if they can offer the same equipment in a reduced size and compact footprint.
2. Will you be able to upgrade the system in case of increased volume requirements?
3. What are the required levels of speed, accuracy, volume and precision?
4. How many plates need to be processed every week?
5. In case of an automated system, how versatile and user-friendly is the associated software?
6. In case of a manual liquid-handling equipment, how ergonomic is the device?

Types of Liquid Handling Devices

1. Manual Pipettes: A manual pipette is a handheld device for accurately measuring and dispensing low liquid volumes. Manual pipettes find application in the field of life sciences, biotechnology and pharmaceuticals.
2. Electronic Pipettes: Electronic pipettes consist of a motor component, which is used for regulating aspiration and rate of dispensing in order to reduce the formation of air bubbles and prevent barrel contamination. In addition, using electronic pipettes minimize measurement errors in studies, wherein pipetting is performed repeatedly as these pipettes are often programmable.
3. Bottle Top Dispensers: A bottle top dispenser is used to dispense highly specific quantities of liquids (generally between 1ml and 100 ml), including chemicals, solvents and oils from bottles or similar containers. Using a bottle top dispenser can help in reducing loss of reagents and increasing efficiency.
4. Pipette Tips: A pipette tip is a disposable and autoclavable cone-shaped attachment of a pipette, which facilitates the transfer of liquids. Pipette tips are available in a variety of formats, including pre-sterilized, non-sterile and filtered tips.
5. Burettes: Burettes are graduated glass tubes, which are used to dispense small liquid volumes, dropwise, especially in titration experiments. The long, glass tube has a tap at its end, used to carefully add liquid drops to a solution. Burettes are available both in manual and digital formats.
6. Pipette Fillers: Pipette fillers are lab instruments used to fill and drain pipettes by suction / pressure. They help in filling and dispensing liquids from pipettes in an extremely controlled manner.

Automated Liquid Handlers: Presently, lab workflows are highly complex and time intensive. At times, the operations involve very minute reagent volumes, and therefore, the quality of liquid transfer needs to be controlled efficiently. Due to this, automated systems have become extremely popular, as they can be optimized for a number of techniques, such as nucleic acid preparation, next-generation sequencing, TLC spotting, ELISA, PCR setup and liquid–liquid extraction.

You need to evaluate the degree of automation required for your operations to run seamlessly. In an automated liquid handler, software applications control almost all the operations of the device. Certain liquid handlers come with pre-scripted protocols for particular assays, and users are able to adjust the settings as required. One can also hire staff having advanced programming skills to design customized protocols.

You should also assess if the budget can be extended to include a semi / full automation system. This can drastically reduce the cost of reagents and operator hours.

Liquid Handling Applications

Liquid handling equipment is used across a wide variety of biological applications, such as molecular biology, drug discovery and development, forensics and materials science.

• Drug Development

Recent advancements in the domain of drug development are coupled with advances in liquid handling equipment and protocols. Various laboratories are now moving away from manual operations and integrating automation in their workflows through the use of high throughput workstations.

“Automation is critical to the modern drug discovery laboratory because it increases the speed and accuracy of tests, saves time, and allows the scientist to focus on science and not manual tasks. The ability to do half a million test points in one day has opened up new strategies in exploring drugs”, states Kevin Hrusovsky, CEO of US-based company, Caliper Life Sciences.

• Food and Beverage Analysis

Liquid handling is critical not only in assessing the nutritional value of food and beverages, it is also fundamental to testing toxins and contaminants present in them.  Various manufacturers have recently opted for electronic pipettes to accelerate their laboratory workflows. Certain laboratories also use liquid handling equipment for detection of food-borne viruses.

• Sample Preparation for Mass Spectrometry

Mass Spectrometry experiments have been transformed completely by the use of robotic liquid handling systems, which have significantly altered the way MS samples are prepared. These systems have allowed laboratories to work smarter and quicker. As per a recent report published in the journal Analytical Sciences, robotic liquid handling systems have the ability to enhance the throughput of an experiment by almost 100 samples.

• Next-generation sequencing (NGS)

Automated liquid handling systems have offered scientists a faster, easier and cost-effective way to prepare NGS samples, as well as sustain the desired high-throughput workflow. Robotic liquid-handling devices greatly enhance the capacity of NGS workflows to run independently with improved performance and precision.

“Automatic liquid-handling systems have the potential to significantly optimize genome sequencing outputs, both in time and costs. As the needs of biological laboratories become clearer, the properties of these pipetting robots also evolve,” according to a report published in the journal BMC Genomics.

Liquid handling Trends

Currently, manufacturers are focusing on using semi-automated systems to enable automation of labs with limited budgets that don’t permit end-to-end automation. Such systems operate through push buttons, thus offering a higher level of ease-of-use and flexibility than a manual pipette. In addition, Artificial intelligence (AI) is changing the way modern labs function. The current focus is on enhancing process security features in order to fulfill the increasing regulatory requirements. Such software updates usually need to be tweaked for appropriate customization of protocols without any compromise in capability.

The primary limitations associated with liquid handling systems are reliability and reproducibility. The devices need to be efficient to function well in difficult experimental conditions with minimal downtime and maintenance requirements. One needs to be familiar with the principles, strengths and weaknesses of the equipment in order to prevent undesirable results.

RNA-sequencing (RNA-seq) is a scientific assay that is used to quantify and examine RNA in a biological sample using next-generation sequencing techniques. The process analyzes the transcriptome to identify the state of genes in our DNA, whether they are turned on or off, as well as the degree of gene activation. Let’s take a look at the working principle of this technique, along with its various applications and challenges.

Working Principle of RNA Sequencing

At the outset, RNA sequencing was performed using Sanger sequencing technology, which is not a cost-and time effective technique in current times. Recently, with the advent of next-generation sequencing (NGS) technology, this assay can be performed at low costs without compromising on the overall throughput.

There are several steps involved in an RNA-seq workflow, including:

• RNA extraction
• Reverse transcription into cDNA
• Adapted ligation
• Amplification
• Sequencing

1) After the extraction of RNA from a sample, the first step is the conversion of RNA of interest into complementary DNA (cDNA) fragments to form a cDNA library. This step is performed through reverse transcription.

2) This is followed by fragmentation of cDNA, and addition of adapters to these fragments. The adapters consist of functional elements that enable the process of sequencing. For instance, the priming and amplification element. After this step, processes, such as amplification, size selection and clean-up are performed.

3) Then, the cDNA library is examined using NGS, which produces short sequences corresponding to the respective fragment.

The depth of RNA sequencing can vary according to the output data requirements. Sequencing can be performed by either single-end or paired-end sequencing techniques. Single-read sequencing is cheaper and faster as the cDNA fragments are sequenced from only one of the ends. In the paired-end technique, sequencing is performed from both ends, but can prove to be beneficial in post-sequencing data reconstruction.

RNA-seq versus Microarrays

RNA-seq is a highly sophisticated technique and is considered to be relatively better than other technologies, including microarray hybridization. The possible reasons for the superiority of RNA-sequencing over other techniques, include:

1) RNA sequencing is not limited by previously identified genomic sequences. Hybridization-based approaches usually require species specific probes, while RNA sequencing is able to identify novel transcripts from entities which have not been sequenced before.

2) The isolated cDNA sequences can be mapped directly to the specific regions in the genome, making the process less erroneous by eliminating the background signal.

3) In microarray experiments, there can be problems related with cross-hybridization or sub-standard hybridization, which are not a major concern in RNA sequencing experiments.

4) Unlike microarray data results, which are relative values calculated with respect to background signals, RNA sequencing data is absolute and quantifiable.

5) Microarrays are ineffective for detection of extremely high or low transcription levels, which is not an issue with RNA sequencing.

Applications of RNA Sequencing

RNA sequencing is useful in examining and discovering various aspects of the transcriptome, such as the mRNA, rRNA and tRNA content. Unraveling the transcriptome can help us co-relate the information stored in the genome with the functional protein expression. In addition, it has the ability to reveal what genes are activated in the genome, as well as their degree of transcription. These discoveries enable researchers to comprehend a cell’s biology in a better way and identify factors that lead to disease. The most common techniques that use RNA sequencing include single nucleotide polymorphism (SNP) identification RNA editing, differential gene expression analysis and transcriptional profiling.

Challenges and Future Perspectives

Although there have been significant strides in the field of RNA sequencing in the last few years, there are various challenges that remain unaddressed.

RNA sequencing requires isolation of sufficient and high-quality RNA, which leads to disposal of a significant portion of a sample. While the sample quantity requirements have minimized substantially over the years, one needs to ensure that they have isolated sufficient amounts of RNA that can fulfill all their data analysis needs. Poor quality samples usually give unsatisfactory outcomes.

Further, RNA degrades rapidly, therefore, it is vital to handle RNA samples with utmost caution at every step of the process. In order to evaluate a sample’s quality and concentration, UV-visible spectroscopy techniques are used.

In an effort to reduce sequencing costs and enable sequencing of small samples, scientists pool samples before the library preparation step. However, while analyzing data, researchers need to account for this factor, and if there are variations within the pooled samples, it can generate misleading results and cause statistical issues.

Over the decade, there has been significant advancement in the domain of RNA sequencing, which is validated through increased throughput and reduced costs. Currently, the sequence fidelity in the process is much better than earlier available NGS technologies. This is accompanied by wider availability of data analysis tools. However, sequencing technologies still need to be improved in order to obtain sufficient read depth and maximize sequencing capacity to increase the number of biological replicates that can be evaluated to generate meaningful data.

As per estimates, around 46.5 million surgical and medical procedures are performed in the US every year. All the procedures are performed using sterile medical devices or equipment to eliminate any sort of contamination. For ensuring the safety of such devices/instruments, procedures like disinfection and sterilization are essential prior to use.

In the past, many studies have reported lack of compliance in terms of established guidelines for disinfection and sterilization, resulting in devastating outbreaks. In this regard, it is important that the healthcare, clinical and hospital authorities establish clear guidelines related to the decontamination mechanism of medical instruments, and whether they need to be cleaned, disinfected or sterilized. These terms, although similar, differ from each other in certain ways. Below is a summary of some key differences.

Decontamination

It is an umbrella term used to describe an item or an environment, which is free from micro-organisms, and therefore safe to handle and use. The terms sterilization, disinfection and cleaning are different forms of decontamination.

1) Sterilization: The process involves killing of all living micro-organisms including spores. However, toxic substances (pyrogens) and dead microorganisms may continue to persist on the surface of the object. In addition, it is important to note that sterilization is not effective against prions.

Degree of sterilization is calculated using the Sterility Assurance Level, which is the probability of survival of a microorganism after the treatment, which should be less than one in one million.

In a laboratory environment, steam sterilization (also known as autoclaving) is often used to achieve optimal levels of sterilization. Autoclaving is performed with saturated steam under pressure of around 15 psi and at least a temperature of 121°C for a certain period of time. This temperature and pressure setting can rapidly destroy microorganisms and decontaminate any other infectious waste. It is often employed to sterilize laboratory glassware and reagents. For an effective heat transfer using autoclaving, the following points must be considered:

• The steam should be able to flush out the air present in the autoclave chamber.
• One needs to check and clean the drain screen present at the bottom of the autoclave chamber. If the drain is blocked, it might result in entrapment of air, which would prevent optimum functioning, and is therefore, critical to achieving sterility.
• In addition, one needs to ensure that the material to be sterilized is in contact with the steam.
• Chemical indicators, such as an autoclave tape should also be used with each batch, which can be a useful indicator of efficacy. Autoclave’s sterility levels must also be monitored on a regular basis through suitable biological indicators, such as B. stearothermophilus spore strips, which are placed at different locations inside the autoclave.
• As spores are highly resistant to heat, each autoclaved container should be spore tested before use.

2) Disinfection

Disinfection is a process of eliminating or destroying the majority, but not all viable organisms. The objective is to minimize the number of microbes to a degree at which they render harmless. However, unlike sterilization, disinfection does not necessarily eliminate spores. Although chemical sterilants have the ability to kill spores with prolonged exposure (3-12 hours).

On the other hand, certain kinds of disinfectants, also known as high-level disinfectants are able to kill all kinds of microorganisms except bacterial spores at a similar concentration as chemical sterilants but with shorter exposure time. In addition, low-level disinfectants are able to kill most vegetative bacteria, certain fungi and viruses, and intermediate-level disinfectants can kill vegetative bacteria, mycobacteria, most fungi and viruses, but not necessarily bacterial spores.

Classification of Disinfection

1. High Level Disinfection

• This level of disinfection requires high concentrations of chemical germicides, such as concentrated sodium hypochlorite.
• High level disinfectants are usually used for shorter periods of time (10-30 min) for disinfection, however, they are able to achieve sterilization if they are in contact with the surface for longer periods of time (7-10 hours).
• This level of disinfection eliminates vegetative microorganisms and inactivates viruses.
• It should not be used on environmental surfaces, such as lab benches or floors.

1.  Intermediate Level Disinfection

• EPA-approved tuberculocidal hospital disinfectants fall into this category.
• It kills vegetative microorganisms and fungi and renders most viruses inactive.
• It can be used for disinfecting lab benches, as well as housekeeping. 

1. Low Level Disinfection

• It does not kill M. tuberculosis
• Eliminates most vegetative bacteria, certain fungi, and inactivates some viruses.
• They are also called ‘hospital disinfectants’ or ‘sanitizers’.

Certain factors, such as prior cleaning of the item, existing organic and inorganic load, type and degree of microbial contamination, physical attributes of the item, biofilm concentration, temperature and pH of the process are crucial to the efficacy of both disinfection and sterilization. These factors must be monitored and evaluated periodically to attain optimal levels of decontamination.